Blotting analysis of wild type (WT) HaCaT and p53 knockdown HaCaT cells

Published: 8 April 2022| Version 1 | DOI: 10.17632/wjhtzjyy45.1
Contributor:
Daniil Romashin

Description

Here we present the experimental data of blotting and immunosorbent analysis of wild type (WT) HaCaT and p53 knockdown HaCaT cells. Provided results were obtained to confirm the knockdown of p53 in HaCaT keratinocytes. HaCaT cells were obtained from Bank of DKFZ, Heidelberg and cultured in DMEM/F:12 medium (1:1, Gibco, USA) supplemented with 1% GlutaMAX (Thermo Fisher Scientific, USA), penicillin/streptomycin (100 UI/mL and 100 μg/mL, Gibco, USA) and 10% v/v fetal bovine serum (Gibco, USA). Cells were grown in 60 mm Petri dishes (Corning, USA) and harvested by trypsinization. The culture medium was replaced by fresh every other day. The knockdown was performed using shRNA designed to knockdown TP53 expression. To perform the knockdown, we applied lentiviral particles (LVPs) encoding anti-TP53 shRNA pLKO-p53-shRNA (Addgene, #25637). To assemble the LVPs, the pLKO-p53-shRNA was delivered in HEK293T cells using PEI transfection. The condition medium was collected 48-72 hours post transfection and purified from cell debris via centrifugation (45 min at 4500g and 4° C). HaCaT cells were grown until 70% confluence, then the cultivation medium was replaced with condition medium containing LPVs for 1.5 hours. The cells were cultured for 4 days under standard conditions, afterwards the medium was replaced with puromycin-containing medium (1 µg/ml). As a control, we used HaCaT cells transduced with lego-ig2 (Addgene, #27341), which does not contain the puromycin resistance gene. After 10 days of puromycin selection 100% of control cells were dead. The obtained cells were used for further experiments. For the immunosorbent assay, the cells were seeded at density of 1×104 cells per well 48 hours prior analysis. ELISA was performed via Abcam’s assay (ab205713) for semi-quantitative p53 determination in accordance with the supplier's manual. For blotting analysis, the cells were grown until confluence of 70% at 60 mm Petri dishes under standard conditions. The cells were rinsed with DPBS and lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0). Then 4x Laemmli buffer was mixed with the samples to 1x followed by 10 min incubation at 90C. The proteins were separated in 8% sodium SDS-PAGE and transfered to PVDF membrane. The immunodetection was performed with antibodies to human p53 (clone BP-53-12, PrimeBioMed, Russia) and β-actin (Bio-rad, VMA00048). Alkaline phosphatase (AP)-conjugated secondary antibodies (Bio-rad, STAR108A) were used for the immunodetection. The chemiluminescence was detected with DNR LumiBis Gel Imaging System 3.2. The images were obtained using GelCapture software. Here we deposit the results of immunoblotting assay confirming the decrease of p53 level in p53 knockdown HaCaT cells. The provided data includes the results of 3 independent experiments. Also, we present the raw data obtained from the ELISA assay.

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Naucno-issledovatel'skij institut biomedicinskoj himii imeni V N Orehovica

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Western Blot

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