Anti tufm

Treated VCaP cells were washed once with cold phosphate-buffered saline (PBS) and lysed by incubating in radioimmunoprecipitation (RIPA) lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mg/mL leupeptin, 10 mg/mL aprotinin, 2 mM NaVO₄, 10 mM β-glycerophosphate, and 1 tablet protease inhibitor) for 1 h on ice. The lysates were centrifuged at 14,000 rpm for 30 min at 4 °C, and the supernatants were collected. The protein concentrations were determined using an enhanced BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Equal amounts of total protein from each sample were resolved by SDS-PAGE on 10% gels and transferred to PVDF (polyvinylidene difluoride) membranes.
After blocking with 50% Odyssey Blocking Buffer in PBS containing 0.05% Tween-20 (PBS-T) for 1 h at room temperature, the membrane was incubated with anti-TUFM (Thermo Fisher Scientific, Rockford, IL, USA), anti-OXCT1 (Novus Biologicals), anti-ACPP (Novus Biologicals), or anti-LDHB (Abcam) primary antibody overnight at 4 °C. The membrane was then washed four times with PBS-T for 5 min each and incubated with the appropriate secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Specific protein bands were detected using the ECL^TM Prime western blotting detection reagent (GE Healthcare Life Sciences).