Horseradish peroxidase hrp conjugated secondary antibody

The concentration of testicular total proteins obtained above was measured using a bicinchoninic acid (BCA) protein assay kit (Beyotime, Nantong, China). About 50 µg of protein from each sample (n = 3 for each group) was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto immuno-blot polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was then blocked in tris-buffered saline with tween 20 (TBST) buffer containing 5% milk for 2 h at room temperature and incubated overnight at 4 °C with primary antibody using the dilutions listed in Table 1. After 1 h of rewarming the next day, the blots were then incubated with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000, CWBIO, Beijing, China). The bonded proteins were visualized with a chemiluminescent HRP substrate (Millipore, MA, USA) using a Universal Hood II Electrophoresis Imaging Cabinet (Bio-Rad, Milan, Italy), and quantified using Quantity One 4.62 software (Bio-Rad, Milan, Italy).

Rabbit polyclonal serum against SetA was produced by Jiaxuan Biotech Company (Shanghai, China). Antibody-containing serum was further affinity-purified against SetA covalently coupled to an Affigel matrix (Bio-Rad) using standard protocols^{48 (link)}. For immunoblotting, the protein samples were separated by SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking with 5% milk for 1 h, membranes were incubated with the appropriate primary antibodies: anti-SetA (Jiaxuan Biotech, China, 1:200,000), anti-FLAG (Cwbio, China, 1:2500), anti-HA (Cwbio, China, 1:2500), anti-His (Cwbio, China, 1:2500), anti-GDI1 (abcom, China, 1:2500), anti-ICDH (Serum specific for Bacillus subtilis ICDH was generously provided by A. L. Sonenshein, Tufts University Medical School, Boston, MA and was used at 1:10,000) overnight at 4 °C. Then the membranes were washed 4 times with Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST), and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Cwbio, China, 1:5000) for 2 h at room temperature. After washing four times with TBST, antibody bands were visualized with the enhanced chemiluminescent (ECL) reagents (Tanon, China) by using a Tanon-5200 Image System (Tanon, China).

Antibodies targeting AKT, phospho‐AKT (P‐AKT Ser473), S6, phospho‐S6 (P‐S6 Ser240/244), GSK3β, phospho‐GSK3β (p‐GSK3β Ser9), β‐catenin, uPA, β‐tubulin, phospho‐β‐catenin (P‐β‐catenin Ser33/37/Thr41), phospho‐B‐Raf (P‐B‐Raf Ser445), phospho‐c‐Raf (P‐c‐Raf Ser338), phospho‐MEK1/2 (P‐MEK1/2 Ser217/221) and P85 were purchased from Cell Signaling Technology (Danvers, MA, USA). β‐actin antibodies were from CMCTAG (Milwaukee, WI, USA), and GRP78 and histone H3 antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies targeting B‐Raf, ERK1/2 and phospho‐FAK (P‐FAK Y397) were purchased from Abcam company (Cambridge, MA, USA). Antibodies targeting MMP2, MMP9, MEK1/2, RAS, c‐Raf, Lamin B, FAK and phospho‐ERK1/2 (P‐ERK1/2 Thr202/Tyr204) were from Wanleibio (Shenyang, China). Active Ras antibody was from NewEast Biosciences (King of Prussia, PA, USA). Horseradish peroxidase (HRP)‐conjugated secondary antibodies were from CWBIO (Peking, China).