Immunoprecipitation, SDS-PAGE, and Immunoblot Analysis

Immunoprecipitation, SDS-PAGE and immunoblot analysis were performed according to a standard protocol as described previously (35 (link)). The following antibodies were used according to the manufacturers’ protocols: anti-Fbxw7 (Abcam, ab12292, RRID: AB_442966), anti-Lys48-specific linked polyubiquitin (MilliporeSigma, 05-1307, RRID: AB_1587578), anti-c-Jun (CST, #9165S, RRID: AB_2130165), anti-p65 (CST, #8242, RRID: AB_10859369), anti-p-p65 (CST, #3036, RRID: AB_331281), Collagen-I(Abcam, ab34710, RRID: AB_731684), Collagen-III (Abcam, ab7778, RRID: AB_306066), EGR-1 (CST, #4154S, RRID: AB_2097035). The PVDF membranes were incubated with corresponding primary antibodies followed by horseradish peroxidase-linked secondary antibodies. The images were developed and captured using Chemiluminescence imaging system (ChemiScope 3000 Mini, Clinx Science Instruments Co., Ltd). Relative quantities of target protein were determined comparing to β-actin expression using densitometric analysis (ImageJ 1.52v). The standard deviation was calculated for biological duplicates.

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He J., Du Y., Li G., Xiao P., Sun X., Song W., Lai L., Xia M., Zhang J, & Wang Q. (2022). Myeloid Fbxw7 Prevents Pulmonary Fibrosis by Suppressing TGF-β Production. Frontiers in Immunology, 12, 760138.

Publication 2021

ab12292 ab34710 ChemiScope 3000 Mini

Actin Antibodies Biological Chemiluminescence Collagen Collagen i Densitometric Egr 1 Fbxw7 Horseradish peroxidase Immunoblot analysis Immunoprecipitation Membranes Polyubiquitin Protein target Pvdf Sds page

Corresponding Organization : Shaoxing University

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Variable analysis

independent variables

Antibodies used: anti-Fbxw7, anti-Lys48-specific linked polyubiquitin, anti-c-Jun, anti-p65, anti-p-p65, Collagen-I, Collagen-III, EGR-1

dependent variables

Relative quantities of target proteins determined by densitometric analysis (ImageJ 1.52v) comparing to β-actin expression

control variables

Experimental procedures performed according to a standard protocol as described previously (35)
PVDF membranes incubated with corresponding primary antibodies followed by horseradish peroxidase-linked secondary antibodies
Images developed and captured using Chemiluminescence imaging system (ChemiScope 3000 Mini, Clinx Science Instruments Co., Ltd)
Standard deviation calculated for biological duplicates

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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