Ecl chemiluminescence system

Freshly isolated hmoDCs were previously pre‐incubated with Pepinh‐MYD or Pepinh‐control for 6 h and with Gefitinib or DMSO for 30 min before the stimulation with control or MV130 for 30 min. Cell lysis was carried out with PBS/Triton 1% in the presence of 1 mM PMSF (Sigma), 1 μg/mL Leupeptin (Bachem) and Aprotinin (Roche) for 30 min at 4 °C with vortex every 10 min. Lysates were clarified by centrifugation at 10 000 × g for 15 min at 4°C. Protein quantification was performed with Micro BCA Protein Assay Kit (Pierce) according to the manufacturer's instructions and samples with equal amounts of total protein were resolved in 10% SDS‐PAGE. Proteins were then transferred to nitrocellulose membranes.
The membranes were incubated with the following antibodies: anti‐human phospho‐IkBα (Ser32/36) (1/1000, Cell signalling), anti‐human phospho‐IKKα/IKKβ (Ser176/Ser177) (1/1000, Cell signalling), and anti‐human β‐actin (1/5000, Sigma) as primary antibodies and Goat anti‐mouse or anti‐rabbit conjugated with horseradish peroxidase (1/2500, Pierce) as secondary antibodies. Reactive bands were visualized by using ECL chemiluminescence system (BioRad). Optical density of the reactive bands was quantified with Fujifilm multigauge software and values expressed relative to β‐actin loading control.

Antibodies against CyclinD1, E-cadherin, β-catenin, Snail1 and GAPDH were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against USP42, CyclinE1, PCNA, and MMP-9 were from Abcam (Cambridge, MA, USA). Anti-MMP-2 was from Epitomics (Burlingame, CA, USA). Horseradish peroxidase-conjugated secondary antibodies were from Beyotime Biotechnology (Shanghai, China).
Cells were washed three times with PBS and then lysed in pre-cooled radioimmunoprecipitation (RIPA) assay buffer on ice for 10 min. After removal of cell debris by centrifugation (12,000g, 10 min), protein concentration of supernatants was measured by BCA protein assay kit (Thermo Fisher Scientific). After boiling for 5 min in sample buffer, equal amount of proteins of different groups were separated by SDS–PAGE, and transferred onto to a nitrocellulose membrane (Millipore, Bredford, USA). After blocking with 5% skim milk, the membranes were incubated with the primary antibodies at 4°C overnight with agitation, followed by incubation with corresponding secondary antibodies for 1 h at room temperature with agitation. Reactive protein was then detected using ECL chemiluminescence system (Bio-Rad, Richmond, CA, USA).

Cells were washed with cold phosphate buffer saline (PBS) twice and lysed with RIPA lysis buffer containing protease and phosphatase inhibitor cocktail (Thermo Scientific™ Halt). After centrifugation of cell lysates, proteins in supernatants were separated by SDS‐PAGE, transferred to polyvinylidene difluoride (PVDF) membrane, blocked in 5% bovine serum albumin (BSA), and incubated with anti‐slc44a1 polyclonal antibody (1:1000, Proteintech), anti‐Caspase‐1 (1:1000, ab179515), anti‐NLRP3 (1:1000, ab263899), anti‐IL‐1β (1:1000, ab283818) overnight at 4°C. The secondary antibody (1:3000, Beyotime) was added to incubate with protein samples for 1 h, and detection was performed by the ECL chemiluminescence system (Bio‐Rad, Hercules).

The protein expression of β-catenin, snail1, a-SMA and podocin in cells or tissues was assayed using western blot analysis. Total protein of MPC5 cells or tissues was extracted in RIPA lysis buffer and then quantified by BCA assay (Thermo Fisher Scientific, Waltham, MA, USA). A total of 50 μg protein was separated by 10% SDS-PAGE and then electro-transferred to polyvinylidene difloride membrane. The membrane was then blocked with TBST containing 5% nonfat dry milk at 4 °C overnight. β-catenin (1:1000, ab32572; Abcam, CA, USA), snail1 (1:1000, ab53519), α-smooth muscle actin (α-SMA) (1:1000, ab5831), and podocin (1:1000, ab50339) were used as the primary antibodies. After washing three times with TBST, the membrane was incubated with appropriate secondary antibodies (1:1000; Abcam, CA, USA). Protein bands were visualized using the ECL chemiluminescence system (BioRad, Hercules, CA, USA). β-actin was set as the internal control.

Protein concentrations were measured by using a Pierce BCA Protein Assay Kit (Thermo Scientific). Most experiments were done with 40 μg protein samples, although we loaded 60 μg of protein for studies related to EGFR in SUM190 cells and 60 μg protein to study E-cadherin in BCX010 cells. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene fluoride membrane by using a semi-dry transfer unit (both from Bio-Rad, Hercules, CA, USA). After being blocked in Tris-buffered saline containing 5% non-fat milk and 0.1% Tween-20, the membranes were incubated with primary antibodies for 2 h at room temperature, and subsequently probed with HRP-conjugated secondary antibodies at room temperature for 1 h. Immunoreactivity was visualized with the ECL chemiluminescence system (Bio-Rad).

For in vitro cell line samples, optimized density of tumor cells was seeded prior to treatment. After being treated for the indicated time and concentration, cells were rinsed with ice-cold PBS and lysed in RIPA buffer with proteases and a phosphatase inhibitor (78440, Thermo Fisher Scientific). Denatured cell extracts were subjected to SDS-PAGE gel electrophoresis before being transferred to PVDF membranes. Blots were blocked and incubated with anti-GCH1 antibodies (ab236387, Abcam) or anti-GAPDH (GB11002, Servicebio, China) for 12–16 hours at 4°C, followed by incubation with appropriate horseradish peroxidase-conjugated antibodies at room temperature for 1 hour. Immunoblotting was detected by the ECL chemiluminescence system (Bio-Rad).

Protein expression levels were determined using western blotting, as previously described31 (link). In brief, total cellular proteins were extracted using RIPA Lysis Buffer (Pierce Biotechnology, Meridian Rd, Rockford, USA). BCA reagents (Pierce Biotechnology, Meridian Rd, Rockford, USA) were used to measure protein concentrations. Equal amounts of proteins (500 μg) were separated by 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The blots were immunoreacted with appropriate primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies, and the proteins of interest were visualized by the ECL chemiluminescence system (Bio-Rad). All the gels were run under the same experimental conditions.