Hrp anti rabbit igg

The levels of the BRF1 protein were evaluated by western blotting. Cells were lysed in RIPA lysis buffer (50 mm Tris-HCl, pH 7.2, 150 mm NaCl, 5 mm EDTA, 1% [wt/vol] (Thermo Scientific)) containing Phosphatase Inhibitor Cocktail (Pierce), and then cleared by centrifugation (15,000×g for 10 min). The protein lysates were mixed with 5 μl of NuPAGE LDS Sample Buffer 4X (Invitrogen) as well as 1M Dithiothreitol (DTT; Sigma) and heated at 70°C for 10 minutes. Denatured samples were resolved on an ExpressPlus PAGE Bis-Tris 4-20% gel (GenScript) and transferred to a nitrocellulose (NC) membrane (Millipore). The membrane was blocked with 5% nonfat dry milk, washed in Tris-Buffered Saline with 0.1% Tween 20 buffer (TBST), and probed with primary antibodies against BRF1 (1:1000 dilution; Rabbit anti-BRF1 from Bethyl) or actin (control) (1:1000 dilution, Mouse anti-actin from Santa Cruz Biotechnology). After washing in TBST, the membrane was incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology anti-mouse BP-HRP 1:5000; or BioLegend HRP anti-rabbit IgG 1:2000). For detection, enhanced chemiluminescence was carried out using the ECL Plus kit (Amersham Biosciences Corp). Protein band quantification was performed using the Image Lab Software v.6.0.1.

The cells were harvested, washed, and lysed with RIPA lysis buffer containing a protease inhibitor cocktail (BBI Life Sciences, Shanghai, China) together with NaF and Na₄P₂O₇, and the protein concentration was measured using a BCA Protein Assay Kit (Pierce, Waltham, MA, USA). Subsequently, the samples were loaded onto SDS-PAGE gels and separated through electrophoresis. The proteins were transferred onto PVDF membranes and incubated with the corresponding primary Abs and HRP-conjugated secondary Abs. Chemiluminescent detection was performed using Alliance 4.7 (UVITEC Cambridge, Cambridge, UK) with Luminata Forte Western HRP Substrate (Millipore, Billerica, MA, USA).
The following antibodies were used: Phospho-IκBα (Ser32) (CST#2859, Cell Signaling Technology, Danvers, MA, USA), IκBα (CST#4812), Phospho-NF-κB p65 (Ser536) (CST#3033), NF-κB p65 (CST#3034), NIK (CST#4994), NF-κB2 p100/p52 (CST#4882), RelB (CST#4922), Phospho-Stat3 (Tyr705) (CST#9131), Phospho-Stat3 (Ser727) (CST#9134), SHP-1 (CST#3759), SHP-2 (CST#3397), Stat-3 (ab68153, Abcam, Cambridge, UK), β-Actin (ACTB) (BM0627, Boster, Wuhan, China), HRP Anti-rabbit IgG (CST#7074), and HRP anti-mouse IgG (BioLegend, 405306).

Protein extracts were obtained by lysing cells in TNN buffer supplemented with 1× Complete ULTRA protease inhibitor (Roche, Basel, Switzerland) and 1× PhosSTOP (ThermoFisher Scientific, Waltham, MA, USA). A total of 25 µg protein was separated on 4–10% Criterion TGX Stain-Free Protein Gels (BioRad, Hercules, CA, USA) or Mini-PROTEAN TGX Stain-Free Protein Gels (BioRad) and immediately transferred to Trans-Blot Turbo Midi PVDF Transfer Packs (BioRad) or Trans-Blot Turbo Mini Transfer Packs (BioRad). The blots were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h at RT followed by incubation with anti-human CDKN2A antibody (Abcam, ab108349, 1:1000), anti-human Mesothelin antibody (Abcam, ab93620, 1:150), anti-human Podoplanin antibody (Abcam, ab236529, 1:500), and anti-human NF2 antibody (Abcam, ab109244, 1:10,000) overnight at 4 °C. The detection antibody HRP anti-rabbit IgG (BioLegend, San Diego, CA, USA, 410406, 1:1000) was incubated for 1 h at RT. Membranes were imaged using either Clarity Western ECL Substrate (Biorad) or SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific) on a Fusion FX (Vilber Lourmat, Eberhardzell Germany). GAPDH and α-Tubulin were used as loading controls, anti-human GAPDH antibody (ThermoFisher Scientific, MA5-15738, 1:1000), and anti-human α-Tubulin antibody (Sigma-Aldrich, T5168, 1:1000).