Ab32043

Proteins were extracted with radioimmunoprecipitation (RIPA) lysis buffer with PMSF, quantified by BCA Protein Assay Kit (Beyotime). Western blot was performed in triplicates according to the procedures reported previously [24 (link)]. GAPDH was used as an internal control. We used the following antibodies: p-AKT (1:2000; rabbit IgG, CST, 4060T), p-ERK1/2 (1:5000; rabbit IgG, Abcam, ab76299), p-STAT5α (1:1000; rabbit IgG, Abcam, ab30648), p-GSK (1:5000; rabbit IgG, Abcam, ab75814), AKT (1:1000; mouse IgG, proteintech, 60203-2-Ig), ERK1/2 (1:1000; rabbit IgG, proteintech, 16443-1-AP), STAT5α (1:1000; rabbit IgG, Abcam, ab32043), GSK3β (1:1000, rabbit IgG, proteintech, 22104-1-AP), CD63 (1:500; rabbit IgG, proteintech, 25682-1-AP), TSG101 (1:500; rabbit IgG, Abcam, ab83), HSP70 (1:100; rabbit IgG, SBI, EXOAB-KIT-1), calnexin (1:2000; rabbit IgG, CST, 2433s), GAPDH (1:10000; rabbit IgG, proteintech, 10494-1-AP) (1:10000; mouse IgG, proteintech, 60004-1-Ig), HRP-conjugated anti-rabbit-IgG (NeoBioscience), HRP-conjugated anti-goat-IgG (NeoBioscience), and HRP-conjugated anti-mouse-IgG (NeoBioscience).

To further reveal the mechanisms of how COL6A1 may influence prostate cancer proliferation, we performed the Cancer Signaling Phospho Antibody microarray PCS248, which was designed and manufactured by Full Moon Biosystems, Inc. (Sunnyvale, CA) and contains 248 antibodies. Each of the antibodies has two replicates that are printed on coated glass microscope slides, along with multiple positive and negative controls. The antibody array experiment was performed by Wayen Biotechnology (Shanghai, China), according to their established protocol. The fluorescence signal of each antibody was obtained from the fluorescence intensity of this antibody spot. A ratio computation was used to measure the extent of protein phosphorylation. The phosphorylation ratio was calculated as follows: phosphorylation ratio = phospho value / unphospho value. Validations were performed by western blot using the following antibodies: anti-JAK2 antibody (ab68269, Abcam), anti-JAK2 (phospho Y221) antibody (ab196009), anti-STAT1 antibody (ab2415), anti-STAT1 (phospho S727) antibody (ab109461), anti-STAT5a antibody (ab32043), anti-STAT5 (phosphoY694) antibody, anti-COL6A1 antibody (17023-1-AP, Proteintech, Chicago, IL, USA), and anti-beta-actin antibody (20536-1-AP, Proteintech, Chicago, IL, USA).