Hrp conjugated secondary antibody

The cells and tissues were lysed with the RIPA buffer (Beyotime) containing 0.1 mmol/L PMSF (Beyotime) on ice for 15 min, and total proteins were isolated by centrifugation at 12,000 rpm for 10 min at 4°C. The concentration of protein extracts was measured by a BCA Assay Kit (Beyotime). Then, the protein extracts were subjected to SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, MA, USA). After blocking with 5% skim milk at 4°C for 4 h, the membranes were immunoblotted with mouse monoclonal antibody against ABCA1 (ab18180, 1:500, Abcam, Cambridge, MA, USA), rabbit monoclonal antibody against ABCG1 (ab52617, 1:1000, Abcam), rabbit monoclonal antibody against CD36 (ab133625, 1:500, Abcam), mouse polyclonal antibody against SR-A (AF1797, 1:1000, R&D Systems, MN, USA), rabbit monoclonal antibody against LXRα (ab176323, 1:500, Abcam), rabbit monoclonal antibody against PPARγ (ab178860, 1:500, Abcam), and rabbit monoclonal antibody against HO-1 (#70081, 1:1000, CST, Danvers, MA, USA) with gentle shaking overnight at 4°C. After a series of rinses with PBST, the membranes were further incubated with HRP-conjugated secondary antibodies (1:5000, CWbio, Beijing, China). The protein bands were visualized with BeyoECL Plus kit (Beyotime) and analyzed using Gel-Pro software 4.0. β-Actin was used as an internal control.

Tumor cell xenografts were cut into 1 × 1 mm pieces on ice and grinded and then scraped into 1 mL of the lysis buffer containing 1% Triton-100, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1 μM pepstatin, 0.5 mg/mL, leupeptin, 0.3 μM aprotinin, 50 mM Tris-HCl (pH 8.0), and 150 mM NaCl. After being lysed on ice for 30 min, the protein samples were centrifuged at 15,000 rpm for 20 min, the supernatants transferred into new tubes, and protein concentration was assayed using the BCA protein assay kit (CWBIO, Beijing, China). For Western blotting, these protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10–15% of SDS-PAGE gels and transferred onto polyvinyldine diflouride membranes (Millipore, Billerica, MA, USA). The membranes were then blocked with 5% bovine serum albumin (BSA) and incubated for 18 h at 4°C with a monoclonal anti-EGFR, anti-phospho-EGFR, anti-MAPK, anti-phospho-MAPK, anti-Akt, anti-phospho-Akt, or anti-β-actin antibody (CWBIO). The next day, the membranes were washed for three times with Tris-based saline-0.1% Tween 20 (TBS-T) and then further incubated for 2 h at room temperature with an HRP-conjugated secondary antibody (CWBIO). Immunoreactive protein bands were further visualized by using the enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA) and exposed to X-ray films.

TM3 cells (10⁶ cells) or 20 mg testicular tissue was added to 100 µl radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) with phenylmethanesulfonyl fluoride (cat. no. ST506; Beyotime Institute of Biotechnology). The concentrations of cell lysates were quantified by a Pierce BCA Protein Assay Kit (cat. no. 23227; Thermo Fisher Scientific, Inc.). Antibodies against Drp1, Mfn1, Fis-1, OPA1, and Bcl-2 were purchased from Abcam (cat. nos. ab184247, ab106274, ab157457, and ab59348). Antibodies against Bax, cleaved caspase 3, and caspase 3 were purchased from Cell Signaling Technology (cat. nos. 5023, 9661, and 9662, respectively). Antibodies against GAPDH were purchased from ProteinTech Group, Inc. (cat. no. 10494-1-AP). HRP-conjugated secondary antibody was obtained from CWBIO (cat. no. CW0103).

Immunohistochemistry staining was performed according to the standard protocol as previous described [23 ]. Polyclonal rabbit primary antibody for Flot2 (Abcam, San Francisco, CA), HRP-conjugated secondary antibody and DAB staining kit (CWBIO, Beijing, China) were used in the experiment. The intensity of staining was scored as 0 (negative), 1 (weak), 2 (medium) or 3 (strong), while the extent of staining was scored as 0 (0% of cell stained), 1 (1–25%), 2 (26–50%), 3 (51–75%) or 4 (76–100%). Then the intensity and the extent scores were multiplied as the final staining scores (0–12) for Flot2 expression. Tumors of final staining score 0–2 were considered as negative, 3–5 as low expression, and 6–12 as high expression.

Tissue samples were fixed in 4% neutral formalin and paraffin-embedded. The tissues were cut into 4-μm thickness and loaded onto the slides. The endogenous peroxidase was blocked with PBS (Sigma–Aldrich Chemical Company, St. Louis, MO, United States) containing 0.3% hydrogen peroxide for a period of 30 min and then with 1% bovine serum albumin (Sigma–Aldrich) for 15 min. The sections were incubated overnight with primary antibody against KI67 (#ab15580, ABcam) at 4°C and then with HRP-conjugated secondary antibody (CW0105, cwbiotech, Beijing, China) at room temperature for 1 h. Finally, immunoreactivity was observed by a diaminobenzidine substrate kit (Thermo Scientific).

Total cellular proteins were extracted from PC-12 cells, using a protein extraction kit (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), and protein concentrations were determined using a BCA protein assay kit (Beyotime P0012S, Shanghai, China) and separated via sodium dodecyl sulphate polyacrylamide gel electrophoresis (15% resolving gel), after which protein bands were electro-transferred onto PVDF membranes (Millipore, MA, U.S.A.). Thereafter, the membranes were blocked in 5% skimmed milk and incubated at 37°C for 1 h and overnight at 4°C with the following primary antibodies (all obtained from Abcam, Cambridge, U.K.): anti-β-actin (rabbit 1:10,000) and anti-VDR (mouse 1:10,00, ab109234). Thereafter, membranes were incubated with HRP-conjugated secondary antibody (1:20,000, anti-rabbit CWBIO, Beijing, China) for 1 h at 37°C, followed by treatment with ECL (Thermo, Waltham, MA, U.S.A.), and the intensity of protein bands was detected using Image Lab™ Software (Bio-Rad, Hercules, CA, U.S.A.).