Hrp conjugated goat anti rabbit igg h l secondary antibody

Frozen testicular tissue was pulverized and homogenized in cold lysis buffer. The homogenate was centrifuged at 12,000
g for 20 min at 4°C, and the supernatant was collected. The protein concentration of the supernatant was measured using the BCA Protein Assay kit (CWBIO, Beijing, China). After being boiled at 100°C for 10 min, samples (50 μg protein/lane) were subject to 10% polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). The membranes were blocked in a 5% non-fat milk in PBST for 2 h at room temperature and then incubated with primary antibodies against HK2 (1:1000 dilution; Cell Signaling Technology), PKM2 (1:1000 dilution; Cell Signaling Technology), LDHA (1:1000 dilution; Cell Signaling Technology), and β-tubulin (1:3000 dilution; Proteintech Group, Inc.) overnight at 4°C. The membranes were washed with PBST and then incubated with HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody (1:5000 dilution; Protein Tech Group Inc) or HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibody (1:5000 dilution; Protein Tech Group Inc.) for 2 h at room temperature. Finally, eECL (CW0049M; CWBIO, Beijing, China) was added, and the Tanon-5500 Chemiluminescence Imaging System (Beijing, China) was used to detect the chemiluminescence of protein bands.

The following antibodies were used in this study: hnRNPF/H (sc-32310, RRID:AB_2248257), Splicing factor, proline- and glutamine-rich (SFPQ) (sc-374502, RRID:AB_10989589), non-POU domain-containing octamer-binding protein (NONO) (sc-166702, RRID:AB_2152178), hnRNPQ (sc-56703, RRID:AB_2200715), hnRNPA2/B1 (sc-374053, RRID:AB_10947257), and glutathione S-transferase (GST) (sc-138, RRID:AB_627677) from Santa Cruz Biotechnology (Dallas, TX); hnRNPM (A500–011A, RRID:AB_11125542) from Bethyl Laboratories (Montgomery, TX); horseradish peroxidase (HRP)-conjugated FLAG (A8592, RRID:AB_439702) from Sigma-Aldrich (St. Louis, MO); HA (11867423001, RRID:AB_390918) and HRP-conjugated HA (12013819001, RRID:AB_390917) from Roche Diagnostics (Basel, Swiss); monoclonal TDP-43 (89789, RRID:AB_2800143) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (2118, RRID:AB_561053) from Cell Signaling Technology (Danvers, MA); β-Tubulin (014–25041, RRID:AB_2650453) from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan); polyclonal TDP-43 (12892–1-AP, RRID:AB_2200505) and polyclonal Matrin3 (MATR3) (12202–2-AP, RRID:AB_2281752) from Proteintech Group (Rosemont, IL); HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (170–6515, RRID:AB_11125142) and HRP-conjugated goat anti-mouse IgG (H + L) secondary antibody (170–6516, RRID:AB_11125547) from Bio-Rad Laboratories (Hercules, CA).

Cells were collected and added appropriate amount of IP lysate (Beyotime, Shanghai, China) for total protein; and nuclear and cytosolic isolation kit (KeyGEN BioTECH, China) for nuclear and cytosolic protein. Equal mass of protein in each sample was separated on SDS‐PAGE and transferred to PVDF membrane (Millipore, Merck KgaA, Darmstadt, Germany). Then, the membrane was blocked in 5% skim milk at room temperature for 1 hour, after that the membrane was incubated with primary antibody (GAPDH, LaminB1, β‐Tubulin, cleaved‐Caspase8, 9, PARP, Bcl‐2, Bcl‐xl, Bad, PCNP, ZEB1, N‐Cadherin, E‐Cadherin were bought from Proteintech, Wuhan, China; AKT, p‐AKT, PI3K, p‐PI3K, GSK3β, p‐GSK3β, β‐catenin were bought from Cell Signaling Technology, MA, USA) at 1:1000 overnight at 4℃. Next, the membrane was incubated with HRP‐conjugated Goat anti‐rabbit IgG (H + L) secondary antibody (1:5000, Proteintech, Wuhan, China) at room temperature for 60min followed by detection of protein band after addition of ECL chemiluminescence (Meilunbio, Dalian, China).