Hrp conjugated anti rabbit antibody

Rat renal biopsies samples and cultured podocyte cells were placed on ice to lyse in cell lysis buffer (Beyotime, Haimen, China) and centrifuged at 12000 rpm for 10 min at 4°C. The total protein in the cell supernatant of each sample was detected using the Bradford method (Pierce, Rockford, IL, USA). An equal amount of protein (25 μg) of each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA). After blocking with 2.5% skim milk for 1 h at 37°C, the members were then incubated with primary antibodies against SOCS2, TLR4, p-p65, p65, p-IκBα, IκBα, Bcl-2, Cleased caspase-3 and β-actin (1:1000; Abcam, Cambridge, MA, USA) at 4°C overnight. The members were then incubated with HRP-conjugated anti-rabbit antibody (1:1000; Santa Cruz, Waltham, MA, USA) for 2 h at room temperature and visualized by using the enhanced chemiluminescence (ECL) system (Pierce).

SET2 or UKE1 cells were treated with either single inhibitor or a combination of PIM inhibitor and JAK2 inhibitor respectively for 4 hours at indicated concentrations. The compounds were then washed off by PBS. The cell pellets were collected and lysed with RIPA buffer (Santa Cruz Biotechnology, Dallas, Texas USA) supplemented with HaltTM phosphatase, protease inhibitor cocktail (Thermo Scientific, Rockford, IL USA,) and phenylmethanesulfonyl fluoride solution (PMSF, Sigma-Aldrich). Lysates were resolved on a 4-12% Bis-Tris gel (Life Technologies, Grand Island, NY, USA) and transferred to nitrocellulose membranes. The membranes were blocked for 1 hour at room temperature with 5% milk, then incubated with the indicated primary antibodies at 1:1000 dilution at 4°C overnight. All primary antibodies were purchased from Cell Signaling (Danvers, MA, USA): Pim1 (2907), Pim2 (4730), Pim3 (4165), c-Myc (5605), pS6-Ser240/244 (2215), S6 (2217), p4EBP1-Thr34/46 (2855), 4EBP1 (9644), pP70S6K-Thr389 (9234), P70S6K (2708), β-actin (5125). After washing, the membranes were incubated with secondary HRP-conjugated anti-rabbit antibody (Santa Cruz) at 1:3,000 for 1 hour at room temperature.

The harvested LNCap, DU145, and PC3 cells were washed with phosphate buffered saline (PBS) and dissolved in lysis buffer. The protein samples were separated using 10% SDS-PAGE and transferred to a 0.45 µM nitrocellulose membrane. After blocking with 5% non-fat dry milk (Rockland), the membrane was incubated at 4 °C with a primary antibody and washed with TBST. The membrane was washed with TBST, incubated at room temperature with a secondary antibody for 1 h, and visualized using an ECL reagent (Thermo Fisher Scientific, San Diego, CA, USA). All experiments were performed in triplicate. Antibodies used in this study are as follows: ANO1 antibody (1:1000) from Santa Cruz Biotechnology, Santa Cruz, CA, USA; ANO8 antibody (1:1000) from Thermo Fisher Scientific, San Diego, CA, USA; ANO10 antibody (1:2000) from Thermo Fisher Scientific, San Diego, CA, USA; HRP-conjugated anti-rabbit antibody (1:5000) from Santa Cruz Biotechnology, Santa Cruz, CA, USA.

10.10⁶ cells were harvested, washed with PBS, and resuspended in lysis buffer (400 mM NaCl, 10 mM HEPES, pH 7.5, 0.1% NP‐40, Sigma P8340); the NaCl concentration was then brought down to 150 mM; and samples were sonicated using a probe sonicator and then centrifuged at 17,000 rcf in order to remove insoluble material. The protein concentration of the lysates was measured using a BCA assay (Thermo Fisher, 23225), and equivalent protein quantities were then incubated with 50 μl of pre‐washed anti‐HA agarose beads (Sigma, A2095) overnight on a rotating wheel at 4°C. The samples were then incubated five times for 10 min with 1 ml of wash buffer (150 mM NaCl, 10 mM HEPES, pH 7.9, 0.1% NP‐40, protease inhibitors) on a rotating wheel at 4°C. Immunoprecipitates were eluted in Laemmli buffer at 95°C for 10 min. Similar elution volumes and protein quantities, for the IPs and the inputs, respectively, were loaded on a gel. Western blots were performed using anti‐Sirt1 (Abcam, ab32441), anti‐IPO7 (Abcam, ab99273), anti‐GFP (Abcam, ab5450), anti‐KAP1 (Merck, MAB3662), HRP‐conjugated anti‐Flag antibody (Sigma, A8592), HRP‐conjugated anti‐HA antibody (Sigma, 12013819001), HRP‐conjugated anti‐goat antibody (Dakocytomation, P0449), HRP‐conjugated anti‐rabbit antibody (Santa Cruz, sc‐2004), and HRP‐conjugated anti‐mouse antibody (GE Healthcare, NA931V).

After separation by SDS‐PAGE, the protein was transferred to a PVDF membrane, and blocked with milk in TBST and then incubated with primary antibodies against rabbit anti‐PD‐L1 (CST, clone E1L3N), p65 (Abcam, clone ab32536) or a rabbit anti‐phospho‐NF‐κB p65 (Ser536) (Abcam, clone ab86299). HRP‐conjugated anti‐rabbit antibody (Santa Cruz Biotechnology) was used as secondary antibodies.

Harvested organs and tumor tissues were embedded in paraffin and divided into several connective sections followed by standard H&E staining protocol. Tissue sections of paraffin-embedded SCC4 and SAS tumors were deparaffinized and rehydrated to retrieve epitopes on the antigens for IHC. Tissue slides were treated with either monoclonal or polyclonal antibodies, such as rabbit polyclonal anti-CD31 (1:100, ab28364, Abcam, Cambridge, MA, USA), rabbit monoclonal anti-Ki-67 (1:200, ab16667, Abcam, Cambridge, MA, USA), and rabbit polyclonal anti-EGFR (1:100, GTX121919; GeneTex, Taipei, Taiwan). Sections of tumor tissues were then incubated with HRP-conjugated anti-rabbit antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 30 min. Slide observation was conducted using a DAB detection kit (Pierce, Rockland, IL, USA). All specimens were examined using an Olympus light microscope (BX53F model, Olympus, Tokyo, Japan). Intensities of IHC stains were further quantified for comparison at 40× magnification (10 images per group) using ImageJ software on 21 July 2016 (version 1.8, National Institutes of Health, Bethesda, MD, USA, http://imagej.nih.gov/ij/).