Goat anti rabbit hrp secondary antibody

Protein lysates were obtained by homogenizing tissue in RIPA buffer (50 mm Tris [pH 7.5], 1 mm EDTA, 1% Triton X‐100, 0.1% sodium deoxycholate, 0.1% SDS, 50 mm NaCl). 25 μg of protein was resolved on 4%–15% acrylamide gels, transferred to PVDF membranes, and probed overnight with primary antibodies: HMBP1 (3935S), p53 (32532S), and tubulin (2146S) from Cell Signaling and p16 (Abcam, Ab51243). Membranes were incubated with goat anti‐rabbit‐HRP secondary antibody (Santa Cruz Biotechnology, sc‐2004) and then ECL was applied to generate signal and imaged with ChemiDox MP imaging system (Bio‐Rad). Intensities of protein detection were quantified using imagej2.

Tissues or cells were homogenized with an ultrasound homogenizer (BANDELIN electronic GmbH) in lysis buffer containing 10% NP40, 0.1 mg/mL n-Dodecyl-β-maltoside, 500 mM sodium fluoride, 10 mM sodium metavanadate, 100 mM PMSF, 1 M Tris (pH 7.5), 0.5 M EDTA and 5 M NaCl, followed by a 60 min incubation on ice to isolate the proteins. Afterwards, the cells were centrifuged and the supernatants were collected and stored at −80 °C overnight. Protein concentrations were determined by the Bradford assay according to the manufacturer’s instructions. Thirty micrograms of protein was separated on a 15% SDS gel and electrophoresed at 80 V. Proteins were transferred to a 0.45 μm nitrocellulose membrane in transfer buffer containing 20% methanol, 0.19 M glycine and 0.025 M Tris (pH 8.3) at 80 V for 2 h on ice. After blocking nonspecific-binding sites with 5% skim milk powder in TBS for 1 h, membranes were incubated overnight at 4 °C with primary antibodies against Mcpt5 (1:200 in 5% BSA in TBS/Tween), and then for 2 h at room temperature with a goat anti rabbit-HRP secondary antibody (Santa Cruz, 1:1,000 in 5% BSA in TBS/Tween).