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Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.


Related in: MedlinePlus

Verification of the BmE75 antibody.(A)Baculovirus-mediated overexpression of BmE75A, BmE75B and BmE75C in Sf9 cells. DsRed2 overexpression was used as a control. After generation of P2 virus, the Sf9 cells were collected and prepared for Western blotting using the BmE75 antibody. (A) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at the initiation of the wandering stage, the fat body was isolated 24 hours after RNAi and prepared for Western blotting using the BmE75 antibody. EGFP dsRNA was used as a control.
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f2: Verification of the BmE75 antibody.(A)Baculovirus-mediated overexpression of BmE75A, BmE75B and BmE75C in Sf9 cells. DsRed2 overexpression was used as a control. After generation of P2 virus, the Sf9 cells were collected and prepared for Western blotting using the BmE75 antibody. (A) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at the initiation of the wandering stage, the fat body was isolated 24 hours after RNAi and prepared for Western blotting using the BmE75 antibody. EGFP dsRNA was used as a control.

Mentions: To understand BmE75 isoforms at the protein level, an antibody was generated against a portion of their common C-termini (Fig. 1B). Both gain-of-function and lose-of-function studies were employed to verify whether the BmE75 antibody was able to simultaneously detect all three isoforms by Western blotting. The three BmE75 isoforms were individually overexpressed in Sf9 cells using the baculovirus-mediated expression system. Western blotting revealed that the molecular weights of BmE75A, BmE75B and BmE75C are 77, 76 and 83 kDa, respectively, in consistent with their predicted molecular weights (Fig. 2A). Importantly, the overexpressed BmE75A, BmE75B and BmE75C have the same molecular weights as their endogenous proteins isolated from the fat body at the wandering stage (Fig. 2B). Moreover, using dsRNA targeting the common region of all three BmE75 isoforms at the initiation of the wandering stage, RNAi-mediated reduction of BmE75 expression lowered the protein levels of all there BmE75 isoforms in the fat body 24 hours after RNAi (Fig. 2B). The above experiments confirmed the authenticity of the BmE75 antibody, which was used throughout the paper.


Bombyx E75 isoforms display stage- and tissue-specific responses to 20-hydroxyecdysone.

Li K, Guo E, Hossain MS, Li Q, Cao Y, Tian L, Deng X, Li S - Sci Rep (2015)

Verification of the BmE75 antibody.(A)Baculovirus-mediated overexpression of BmE75A, BmE75B and BmE75C in Sf9 cells. DsRed2 overexpression was used as a control. After generation of P2 virus, the Sf9 cells were collected and prepared for Western blotting using the BmE75 antibody. (A) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at the initiation of the wandering stage, the fat body was isolated 24 hours after RNAi and prepared for Western blotting using the BmE75 antibody. EGFP dsRNA was used as a control.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4499807&req=5

f2: Verification of the BmE75 antibody.(A)Baculovirus-mediated overexpression of BmE75A, BmE75B and BmE75C in Sf9 cells. DsRed2 overexpression was used as a control. After generation of P2 virus, the Sf9 cells were collected and prepared for Western blotting using the BmE75 antibody. (A) dsRNA targeting a common region of all three BmE75 isoforms was injected into Bombyx larvae at the initiation of the wandering stage, the fat body was isolated 24 hours after RNAi and prepared for Western blotting using the BmE75 antibody. EGFP dsRNA was used as a control.
Mentions: To understand BmE75 isoforms at the protein level, an antibody was generated against a portion of their common C-termini (Fig. 1B). Both gain-of-function and lose-of-function studies were employed to verify whether the BmE75 antibody was able to simultaneously detect all three isoforms by Western blotting. The three BmE75 isoforms were individually overexpressed in Sf9 cells using the baculovirus-mediated expression system. Western blotting revealed that the molecular weights of BmE75A, BmE75B and BmE75C are 77, 76 and 83 kDa, respectively, in consistent with their predicted molecular weights (Fig. 2A). Importantly, the overexpressed BmE75A, BmE75B and BmE75C have the same molecular weights as their endogenous proteins isolated from the fat body at the wandering stage (Fig. 2B). Moreover, using dsRNA targeting the common region of all three BmE75 isoforms at the initiation of the wandering stage, RNAi-mediated reduction of BmE75 expression lowered the protein levels of all there BmE75 isoforms in the fat body 24 hours after RNAi (Fig. 2B). The above experiments confirmed the authenticity of the BmE75 antibody, which was used throughout the paper.

Bottom Line: At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E.At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues.In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

View Article: PubMed Central - PubMed

Affiliation: 1] Guangdong Provincial Key Laboratory of Agro-animal Genomics and Molecular Breeding/Guangdong Provincial Sericulture and Mulberry Engineering Research Center, College of Animal Sciences, South China Agricultural University, Guangzhou, 510642, China [2] Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.

ABSTRACT
Resulted from alternative splicing of the 5' exons, the nuclear receptor gene E75 in the silkworm, Bombyx mori, processes three mRNA isoforms, BmE75A, BmE75B and BmE75C. From the early 5(th) larval instar to the prepupal stages, BmE75A mRNA and protein levels in the prothoracic glands display developmental profiles similar to ecdysteroid titer. In the fat body, mRNA levels but not protein levels of all three BmE75 isoforms correlate with ecdysteroid titer; moreover, proteins of all three BmE75 isoforms disappear at the prepupal stages, and a modified BmE75 protein with smaller molecular weight and cytoplasm localization occurs. At the early 5(th) larval instar stage, treatment of the prothoracic glands and fat body with 20-hydroxyecdysone (20E) and/or cycloheximide (CHX) revealed that BmE75A is 20E primary-responsive at both mRNA and protein levels, while BmE75B and BmE75C exhibit various responses to 20E. At the early wandering stage, RNAi-mediated reduction of gene expression of the 20E nuclear receptor complex, EcR-USP, significantly decreased mRNA and protein levels of all three BmE75 isoforms in both tissues. In conclusion, BmE75 isoforms display stage- and tissue-specific responses to 20E at both mRNA and protein levels; moreover, they are regulated by other unknown factors at the protein level.

No MeSH data available.


Related in: MedlinePlus