Anti gapdh sc 365062

Cells or tissues were lysed in RIPA lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 0.1% SDS, 1% NP-40, 0.25% sodium deoxycholate and 1 mM EDTA, pH 8.0) with freshly added protease inhibitor cocktail (Roche) for 30 min on ice and were then centrifuged at 16,000 × g at 4 °C for 10 min. The supernatant was collected, and the protein concentration was calculated with a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Proteins were separated by SDS-PAGE (Bio-Rad). After electrophoresis, the proteins were electrotransferred onto PVDF membranes (Bio-Rad) and then blocked with 5% skim milk for 1 h. The membranes were then incubated with primary antibodies at 4 °C for 12 h. After three washes in TBST, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. After three washes, the membranes were incubated with the SuperSignal West Pico chemiluminescence substrate (Pierce). The protein levels were normalized by probing the same blots with an anti-GAPDH antibody. The antibodies were purchased from the following sources: anti-LIN28B (F-21): (sc-130802, Santa Cruz Biotechnology, CA, USA) and anti-GAPDH (sc-365062, Santa Cruz Biotechnology, CA, USA). Protein bands were analyzed using the ImageJ software.

The following antibodies were used: anti-OTUB1 (ab175200), anti-OTUB1 (ab198214, for IHC), anti-Smurf1 (ab57573), anti-RunX2 (ab192256), and anti-OSX (ab209484), purchased from Abcam. Anti-OTUB2 antibody (GTX83953) was purchased from GeneTex. Anti-FGFR1 (9740), anti-FGFR2 (23328), anti-FGFR3 (4574), anti-AKT (4691), anti-p-Ser473-AKT (4060), anti-p-Ser308-AKT (13038), anti-ERK1/2 (4695), and anti-p-Thr202/Tyr204-ERK1/2 (4370) antibodies were purchased from Cell Signaling Technology. Anti-GAPDH (sc-365062) and anti-GST (sc-374171) antibodies were purchased from Santa Cruz Biotechnology. Anti-Myc (M047-3), anti-Flag (M185-3), anti-HA (M180-3), and anti-Multi Ubiquitin (D058-3) antibodies were purchased from MBL. Anti-β-actin (AC026) and anti-COL1A1 (A16891) antibodies were purchased from Abclonal. Peroxidase-AffiniPure goat anti-rabbit IgG (111-035-003) and Peroxidase-AffiniPure goat anti-mouse IgG (115-035-003) were purchased from Jackson.
Full-length OTUB1 WT, D88A, C91S, H265A, ASA mutants, and Flag-HECT E3s were cloned into the pFlag-CMV-2 vector. Full-length FGFR2 was cloned into the pEF6/Myc-His vector.

Anti-Rhodopsin (MS-1233-P; Ab-1 (RET-P1); Neomarkers, Fremont, CA, USA); anti-trkA^NGFR (sc-118; Santa Cruz Biotech, Santa Cruz, CA, USA); anti-phospho trkA^NGFR (p-trkA^NGFR; sc-130222; Santa Cruz); anti-p75^NTR (sc-6188; Santa Cruz); and anti-GAPDH (sc-365062; Santa Cruz). Alexa Fluor 488-conjugated goat anti-rabbit and Alexa Fluor 594-conjugated goat anti-mouse as well as horseradish peroxidase-conjugated anti-rabbit/mouse specie-specific secondary antibodies (Cell Signaling Technology, Danvers, MA, USA).

Human HOS, U2OS, SAOS‐2, MG63, SOSP‐9607 and 143B osteosarcoma cell lines, and the human osteoblast hFOB1.19 and NHOst were purchased from Shanghai Ruilu Technology Co. (Shanghai, China). Cells were cultured in Dulbecco's modified Eagle medium (DMEM)/high glucose supplemented with 10% foetal bovine serum (FBS; Gibco, Carlsbad, CA, USA) and 1% penicillin and streptomycin (Invitrogen, Carlsbad, CA, USA). And cells were incubated in a humidified incubator at 37°C with 5% CO₂.
Anti‐GAPDH (sc‐365062), anti‐Vimentin (sc‐ 373717), anti‐N‐cadherin (sc‐393933), anti‐E‐cadherin (sc‐7870), mouse monoclonal antibodies and the horseradish peroxide‐conjugated mouse IgG secondary antibody (sc‐2025) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Two different anti‐human siRNAs of HNF1A‐AS1 and scramble siRNA, purchased from Life Technologies (Carlsbad, California, USA) were used: HNF1A‐AS1 siRNA1 (Cat. no. n265374), HNF1A‐AS1 siRNA2: (Cat. no. n265377). Cells were transfected with HNF1A‐AS1 siRNAs or scramble using Lipofectamine 3000 (Invitrogen) according to the manufacturer's protocol.

Small interfering RNAs (siRNAs) were purchased from Shanghai Genechem Company Limited. Targeting sequence of all siRNAs were listed in Supplementary Table S1. GOT1 expression vector (#HG14196-CM) were obtained from Sino Biological Inc. (Beijing, China). Anti-GDH1 (#ab89967), Anti-GOT1 (#ab170950) were obtained from Abcam Inc. (MA, USA) and Anti-β-actin (#9027) were purchased from Cell Signaling Technology Inc. (BOS, USA). Anti-GAPDH (sc-365062) were obtained from Santa Cruz Biotechnology (TX, USA).