Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Liver lysosomes of fed Snell mice show more uptake of CMA substrates than lysosomes from littermate controls. (A) Representative western blots of liver lysosomes from control (C) and Snell (S) mice 2 h after treatments with leupeptin (100 mg/kg body weight) or PBS vehicle control. (B-D) Quantifications of the abundance of CMA substrates GAPDH, ENO1, and ACADL in lysosomes shown in (A). (E) Quantification of PPID, endosomal microautophagy substrate. (F) Quantification of HSPA8, CMA chaperone. (G) Representative western blots for binding and uptake assays for CMA substrate GAPDH, quantified in (H). (I) Representative western blots for binding and uptake assays for CMA substrate MAPT, quantified in (J). For (B-F), n = 9 of each treatment group, 2-way ANOVA results are reported below each bar graph. Comparisons plotted on the bar graphs are results of Student’s t-test. For (H & J), n = 6 of each treatment group, the results of Student’s t-test are shown on the graphs. Error bars are S.E.M

Article Snippet: Recombinant GAPDH (Sino Biological, 51,221-M07E), MAPT (Sino Biological, 10,058-H07E), and/or protease inhibitors (Sigma, 11,836,153,001) were added, as indicated.

Techniques: Western Blot, Binding Assay

Journal: Autophagy

Article Title: Long-lived mice with reduced growth hormone signaling have a constitutive upregulation of hepatic chaperone-mediated autophagy

doi: 10.1080/15548627.2020.1725378

Figure Lengend Snippet: Liver lysosomes of fed ghr KO mice show more uptake of CMA substrates than lysosomes from littermate controls. (A) Representative western blots of liver lysosomes from control (C) and ghr KO (KO) mice 2 h after treatments with leupeptin (100 mg/kg body weight) or PBS vehicle control. (B-D) Quantifications of the abundance of CMA substrates GAPDH, ENO1, and ACADL in lysosomes shown in (A). (E) Quantification of PPID, endosomal microautophagy substrate. (F) Quantification of HSPA8, CMA chaperone. (G) Representative western blots for binding and uptake assays for CMA substrate MAPT, quantified in (H). For (B-F), n = 6 of each treatment group, 2-way ANOVA results are reported below each bar graph. Comparisons plotted on the bar graphs are results of Student’s t-test. For (H), n = 6 of each treatment group; the results of Student’s t-test are shown on the graphs. Error bars are S.E.M

Article Snippet: Recombinant GAPDH (Sino Biological, 51,221-M07E), MAPT (Sino Biological, 10,058-H07E), and/or protease inhibitors (Sigma, 11,836,153,001) were added, as indicated.

Techniques: Western Blot, Binding Assay

Journal: PLoS ONE

Article Title: Spermatogenesis Associated 4 Promotes Sertoli Cell Proliferation Modulated Negatively by Regulatory Factor X1

doi: 10.1371/journal.pone.0075933

Figure Lengend Snippet: After transient transfection of pcDNA3.1-RFX1 for 36 h, the overexpressed RFX1 protein was detected by immunocytochemistry staining and Western blot ( A ), overexpression of RFX1 was shown to down-regulate the promoter activity ( B ) and Spata4 mRNA expression ( C ) and in a dose-dependent manner. The Spata4 mRNA expression was analyzed by quantitative PCR and normalized to GAPDH. ** P <0.01. *** P <0.001.

Article Snippet: The membranes were then incubated with primary antibodies specific for RFX1 (1∶500; Santa Cruz), Spata4 (1∶1000; prepared by our laboratory), GAPDH (1∶1000; Cwbiotech, Beijing, China) or β-tublin (1∶1000; Cwbiotech) at 4°C overnight, followed by an incubation with horseradish peroxidase-conjugated anti-goat IgG (1∶5000; Santa Cruz) or anti-rabbit IgG (1∶10000) at room temperature for 1 h. The chemiluminescence reaction was performed using ECL reagent (Thermo Scientific).

Techniques: Transfection, Immunocytochemistry, Staining, Western Blot, Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction