Ab195055

HRMECs were collected and extracted by RIPA lysis buffer (89901, Thermo Scientific, U.S.A.) with protease inhibitor cocktail (Merck KGaA, Darmstadt, Germany). A BCA™ Protein Assay Kit (Pierce, Appleton, WI, U.S.A.) was used to evaluate the protein levels. Equal amounts of proteins were loaded on 10% SDS-PAGE gels (Thermo Scientific), transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, Massachusetts, U.S.A.) and blocked with 5% non-fat milk at room temperature for 1 h. Afterwards, membranes were subjected to immunoblotting with primary antibodies (all purchased from Abcam, Cambridge, MA, U.S.A.) against SOCS6 (ab197335, 1/500), p-JAK2 (ab195055, 1/500) JAK2 (ab39636, 1/1000), p-STAT3 (ab32143, 1/1000) STAT3 (ab119352, 1/5000), PCNA (ab92552, 1/1000), Cyclin D1 (ab16663, 1/200), Cyclin E1 (ab33911, 1/1000), cleaved caspase-3 (ab32042, 1/500), Bcl-2 (ab59348, 1/500) and Bax (ab216494, 1/100) and incubated at 4°C overnight. Subsequently, the blots were washed with PBST, and the membranes were incubated with secondary peroxidase-linked goat anti-rabbit IgG H&L (1:5000, Santa Cruz, California, U.S.A.) at room temperature for 2 h. Signals were visualized by the ECL (Amersham Biosciences, Piscataway, NJ) method after washing, while optical densities of the bands were measured using ImageJ software (Bio-Rad).

Total protein extracts were prepared from the harvested testes using radio-immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, St. Louis, MO, USA) and stored at −80 °C. The primary antibodies for JAK2 (ab108596) and ph-JAK2 (ab195055) were purchased from abcam (Cambridge, UK). The primary antibodies for STAT3 (mAb#4904) and ph-STAT3 (mAb#9145) were purchased from Cell Signaling Technology (Danvers, MA, USA). The primary antibodies for Nrf2 (MBS9600480) and Keap1 (MBS2536215) were purchased from MyBioSource.com (San Diego, CA, USA). Protein extracts (150 μg) were resolved through 10% SDS-PAGE. Separated proteins were transferred to a PVDF membrane followed by blocking and incubation with primary antibody for each target protein individually. The primary antibody dilutions are as follows: JAK2 (1:1000), ph-JAK2 (1:500), STAT3 (1:1000), ph-STAT3 (1:500), Nrf2 (1:1000), and Keap1 (1:1000). The PVDF membrane was then treated with a horseradish peroxidase/HRP-conjugated secondary antibody. Protein band signal amplification was performed using the electrochemiluminescence method (ECL) (Thermo Fisher Scientific, Waltham, MA, USA) and visualized by the ChemiDoc™ MP Imaging System (BioRad, Hercules, CA, USA). Band intensity quantification was measured using the Image Lab software v. 4.1 (BioRad, Hercules, CA, USA).

Total protein of cells was extracted using high-efficiency RIPA lysis buffer (R0010, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) in strict accordance with the instructions. The protein concentration was determined using a BCA kit (20201 ES76, Yeasen Company, Shanghai, China). Then, the protein was separated by polyacrylamide gel electrophoresis and then transferred to PVDF membranes by wet transfer method. Next, the membranes were blocked using 5% BSA at room temperature for 1 h and probed overnight at 4 °C with the following diluted primary antibodies: mouse anti-human GAPDH (ab9425, 1:2500, Abcam, Cambridge, UK), JAK2 (ab39636, 1:1000, Abcam, Cambridge, UK), p-JAK2 (ab195055, 1:1000, Abcam, Cambridge, UK), STAT3 (ab31370, 1:500, Abcam, Cambridge, UK), and p-STAT3 (ab86430, 1:500, Abcam, Cambridge, UK). The membranes were washed for three times (5 min/time) with Tris-buffered saline Tween-20 (TBST), and horseradish peroxidase (HRP)-labeled goat anti-rabbit immunoglobulin G (IgG) (ab205718, 1:20,000, Abcam, Cambridge, UK) was added for 1 h of incubation at room temperature. Then, ImageJ 1.48u software (National Institutes of Health) was performed for protein quantification analysis. The ratio of the gray value of the target band to GAPDH was representative of the relative protein expression. The experiment was repeated three times.

For JAK2 inhibition, Tyrphostin AG490 (#3434) was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Antibodies against p-JAK2 (ab195055) and ATM (2CI) were purchased from Abcam (Cambridge, UK) and Santa Cruz Biotechnology (Dallas, TX, USA), respectively. Antibodies against Chk1(LS-C352010), p-Chek1 (LS-C358948), Chk2 (LS-C358943), p-Chk2 (LS-C416418), p-ATM (LS-C353715) were all purchased from Lifespan BioSciences (Seattle, WA, USA). Antibodies against p-STAT1 (#8826) and p-STAT3 (#9145), γH2AX (#9718), ATR (#2790), p-ATR (#2853) and GAPDH (#2118) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies for Western blot: peroxidase-conjugated AffiniPure Goat anti-Rabbit (#133499) and anti-Mouse (#133599) were purchased from Jackson ImmunoResearch (West Grove, PA, USA). For anesthesia, Ketamine and Xylazine were purchased from Hikma Pharmaceuticals (Amman, Jordan) and Bayer GMP (Bergkamen, Germany), respectively.

Western blotting was carried out as previously described [22 (link)]. Briefly, total protein from tissues and cells was extracted by using a total cell protein extraction kit (KeyGen Biotechnology, Nanjing, China). Then, equivalent amounts of protein were separated by SDS-PAGE and transferred onto the polyvinylidene difluoride (PVDF) and blocked with 2% bovine serum albumin (Beyotime Biotechnology, Beijing, China) for 2 h. The membrane were incubated overnight at 4 °C with the primary antibodies: β-actin (1:2000; #20536-1-AP, Proteintech), XPR1 (1:1000; #14174-1-AP, Proteintech), IL6 (1:1000; #ab233551, Abcam), IL6 (1:1000; #ab233551, Abcan), p-JAK2 (1:500; #WL02997, Wanleibio, Shenyang, China), JAK2 (1:500; #ab195055, Abcam), p-STAT3 (1:1000; #WLP2412, Wanleibio), STAT3 (1:2000; #ab76315, Abcam), IGF2BP3 (1:1000; #14642-1-AP, Proteintech), followed washing with PBS/T (Phosphate Buffer Solution with 0.05% Tween20). Next, the membranes were incubated with horseradish peroxidase-linked secondary antibody (1:1000; #SA00001-2, Proteintech) at room temperature for 1 h. Finally, immunoreactive bands were displayed by a chemiluminescence ECL kit (Beyotime Biotechnology, Beijing, China) and quantified by Image J software (National Institutes of Health, Bethesda, MD, USA) while the β-actin was used as internal control.