Trans blot turbo rta transfer kit

SDS-PAGE was performed using Mini-PROTEAN TGX Precast Gels (Bio-Rad) and the Mini-PROTEAN Tetra Cell system (Bio-Rad) at 90V. Transfer of protein from the gel to a PVDF membrane were performed using a Trans-Blot Turbo Transfer System (Bio-Rad), a PowerPac Basic (Bio-Rad) power supply, and a Trans-Blot Turbo RTA Transfer Kit (Bio-Rad). Precision Plus Protein Dual Color Standard (Bio-Rad) was used to monitor electrophoresis and to detect transfer to the PVDF membrane. A biotinylated protein ladder (Cell Signaling Technology, Danvers, MA, USA) was detectable during chemiluminecent imaging.

Cells were lysed in RIPA buffer (Millipore, #20–188). After centrifugation, cell debris was discarded and protein concentration in the supernatant was determined by Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, #23225). Colorimetry was measured using plate reader, Victor X3 (Perkin Elmer). 5 μg of total cell lysates were mixed with Laemmli Sample Buffer (Bio-Rad, #161–0747) and 10% of 2-Mercaptoethanol (SIGMA Aldrich, #M6250) and used for western blotting. Blotting of the proteins to membrane was performed using Trans-blot Turbo RTA Transfer Kit (Bio-rad, #170–4272) as follows. After developed on SDS gels, proteins were transferred on PVDF membranes (Bio-Rad, a component of the Transfer Kit) by using Trans-blot Turbo system (Bio-Rad). Membranes were then blocked with 1% skim milk for an hour, and overnight with the relevant diluted primary antibodies. Then, membranes were washed three times with Tris buffer Tween 20 (TBST) and incubated with diluted secondary antibodies. One hour later, membranes were washed again three times with TBST, then protein-antibody complexes were visualized by Super Signal ELISA Femto Maximum Sensitivity Substrate (Thermo Scientific, #37074).

Cells were harvested in PBS and homogenized in RIPA buffer (ThermoFisher Scientific #89900) supplemented with protease inhibitors (Takara #ST0293), for 10 min at 4°C before centrifugation at 12,000 g at 4°C for 20 min. Proteins were quantified using the Pierce BCA Protein Assay kit (ThermoFisher Scientific #23225). Total protein extract between 30 and 80 μg were loaded in a NuPAGE 4–12%, Bis-Tris, 1.5mm Mini Protein Gel (Bio-Rad #NP0321BOX). Transfer into a PVDF membrane was performed using the TRANS-Blot Turbo RTA Transfer Kit (Bio-Rad #1704272). After transfer, membranes were blocked with a 5% solution of nonfat powdered milk (LabScientific #732-291-1940) in PBS 0.1% Tween for a minimum of 1 h before antibody hybridization. The following primary antibodies were incubated overnight at 4°C: POU2F1 (1:1000) (Abcam #ab178869) and β-actin (1:1000) (Abcam #ab6276). HRP Anti-rabbit (Millipore Sigma #NA934v) and HRP Anti-mouse (Millipore Sigma #NA931v) were used as secondary antibodies for detection using luminescence (Clarity Max Western ECL Blotting Substrates (Biorad #1705062). Images were captured using a Chemidoc MP Image System (Bio-Rad) and cropped using the Image Lab Software. Experiments were repeated using total protein extract from biological triplicates.

Primary monoclonal antibodies (mouse origin)
for HO-1 (sc-390991) and β-actin (sc-8432) were purchased from
Santa Cruz Biotechnology (Texas). The secondary antibody (goat-antimouse
HRP-conjugated IgG) was purchased from Biorad (Hercules, CA). After
treating the cells with specified conditions, the culture medium was
aspirated, and the cells were washed three times with PBS. Cells were
lysed with 120 μL of 1× Laemmli loading buffer (contains
2.5% mercaptoethanol) and denatured at 95 °C for 5 min. 2–5 μL
of denatured cell lysate was loaded onto the 10% SDS-PAGE gel (Biorad),
and electrophoresis was conducted with a Biorad system under constant
voltage (150 V). The protein was transferred to the PVDF membrane
with a Trans-Blot Turbo RTA transfer kit (Biorad). The membrane was
blotted by the iBind Flex system (Thermo Fisher), using HO-1 (1:100),
β-actin (1:400), and HRP-conjugated secondary antibody (1:1500).
The chemiluminescence of the protein band was imaged with LAS4000
(GE Healthcare, Chicago, IL) and analyzed with InteliQuant software.

Proteins were isolated using RIPA buffer (Boston BioProducts, BP-115) with protease inhibitor (Roche, 4693132001) and phosphatase inhibitors (New England Biolabs, P0758L). Protein concentrations were determined using Pierce BCA assay (Thermo Fisher Scientific). A total of 20 μg protein were loaded per lane on a 4%–20% Mini-PROTEAN TGX Gel (Bio-Rad, 456-1096). Separated proteins were transferred to PVDF membranes using the Transfer Turbo Blot system (Bio-Rad) and Trans-Blot Turbo RTA Transfer Kit (Bio-Rad, 170-4272). The membrane was blocked with 5% nonfat milk in TBST for 1hour at room temperature. After blocking, the membrane was incubated overnight at 4°C with antibodies against Flag Tag (Cell Signaling Technology, 2368, 1:1000), GAPDH (Cell Signaling Technology, 2118, 1:4000), VCAM-1 (Cell Signaling Technology, sc-13160, 1:1000), ICAM-1 (R&D Systems, Bio-Techne, BBA3), IκBα (Cell Signaling Technology, 4812, 1:1000), β-actin (Cell Signaling Technology, 4970, 1:3000), and phospho-IκBα (Cell Signaling Technology, 2859, 1:1000), IL-1β (Abcam ab9722, 1:1000), MCP-1 (Abcam ab25124, 1:1000), COX-2 (Cell Signaling Technology 12282p), p-P38MAPK (Cell Signaling Technology 4511L, 1:1000), and P38 MAPK (Cell Signaling Technology 9212L, 1:1000). Quantification of protein bands was performed using a luminescent image analyzer (Bio-Rad, Chemidoc).