Supersignal west pico plus kit

Proteins were isolated from two to three human cortical organoids at 6 weeks or from N2a cells that overexpressed human EREG for 24 h. Briefly, N2a cells were plated in six-well plates and grown in N2a medium containing DMEM/F12, 10% FBS, and 1X penicillin/streptomycin. Cells were transfected with a pCAG-empty or pCAG-EREG plasmid, together with pCAG-GFP plasmid, using lipofectamine 2000 according to the manufacturer’s instructions. Protein concentration was measured using the Pierce detergent-compatible Bradford assay kit (Thermo Scientific, #23246). Subsequently, 40 µg of protein were resolved on a 10% SDS Polyacrylamide gel and transferred to a PVDF transfer membrane (Thermo Scientific, #88518). Membranes were blocked for 1 h at room temperature with 5% skim milk in PBS with 0.05% tween, and then incubated with primary antibodies overnight at 4 °C. Secondary antibodies were incubated for 1 h at room temperature. Antibody signal was detected using the SuperSignal West Pico plus kit (Thermo Fisher Scientific, #34579).

CAOV3 and SKOV3 cells were incubated with 6-ME alone or in combination with NAC or OAA for 24 h. These cells were then collected, incubated with appropriate cell lysis buffer on ice for 20 min, and centrifuged at 12, 000 rpm for 20 min at 4°C. After transferring the supernatants to a new 1.5 ml EP tube on ice, the protein concentration was determined using Pierce™ BCA Protein Assay kit (Thermo Fisher Scientific, 23,225) following the manufacturer’s protocol. Subsequently, the protein concentration was adjusted to 1 μg/μL/sample with 5X DualColor Protein Loading Buffer (Fude Biological Technology, FD006) and heated at 95°C for 5 min in a Digital Dry Baths/Block Heaters (Thermo Fisher Scientific, 88870005). For western blot analysis, protein samples (20 μg/each) were loaded onto the SDS-PAGE gel, electrophoresed, and transferred onto 0.22 μM PVDF membranes. Afterblocking with 5% NON-Fat Powdered Milk (Solarbio Life Science, D8340) for 90 min, the membranes were incubated with the desired primary antibodies overnight at 4°C, washed with 1X TBS-T for 5 min at least three time, and incubated with corresponding secondary antibodies at room temperature for 90 min. Finally, these membranes were washed with 1X TBS-T for 10 min at least three times, visualized with the SuperSignal™ West Pico PLUS kit (Thermo Fisher Scientific, 34,580), and quantified using ImageJ software.

Jejunal and ileal mucosal tissues were lysed in 1x RIPA buffer and equal amounts were separated on 4–12% Bis-Tris NuPAGE gels in MES running buffer (Thermo Fisher Scientific, Germany). After wet blotting in 1x transfer buffer onto PVDF membranes (Merck, Darmstadt, Germany) proteins were blocked by 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 and incubated with an anti–lysozyme-1 antibody (1:2000, abcam, Germany) and 1:2000 HRP-coupled secondary antibody (Cell Signaling Technologies, Germany). Proteins were detected by the Super Signal West Pico Plus Kit (Thermo Fisher Scientific, Germany). Afterwards the membranes were stripped with the Restore^TM Plus Western Blot stripping kit (Thermo Fisher Scientific, Germany) for 15min and incubated with an anti–beta actin antibody (1:25000, Sigma Aldrich, Germany) followed by a 1:200 diluted HRP-coupled secondary anti-mouse antibody (Cell Signaling, Germany).

Adherent A375 and A375R cells were lysed in RIPA buffer (Thermo Scientific) supplemented with 1% protease and phosphatase inhibitors (Thermo Scientific). Protein amount in whole cell lysates was measured with a Pierce™ BCA Protein Assay Kit (Thermo Scientific). Equal amounts of protein were loaded onto 4%‐15% Mini‐PROTEAN^® TGX™ Precast Gels (Bio‐Rad). Following electrophoresis in 1× Tris/glycine/SDS running buffer (Bio‐Rad), proteins were transferred to PVDF membranes using the Trans‐Blot^® Turbo™ RTA Mini PVDF Transfer Kit (Bio‐Rad) according to the vendor's instructions. Non‐specific binding was blocked by soaking the membranes in 5% BSA in tTBS (1× Tris‐Buffered Saline, 0.1% Tween 20, Bio‐Rad) at room temperature for 1 hour.
Membranes were incubated with primary anti‐HSP90, anti‐LDHA, anti–β‐actin, anti‐HSP90, anti–c‐MYC, (Cell Signaling), anti‐MCT1, anti‐MCT4, anti‐GLUT1 (Abcam) antibodies in tTBS‐BSA 5% at 4°C overnight, followed by incubation with anti‐rabbit or anti‐mouse secondary antibodies (Jackson IR) in tTBS‐BSA 1% at room temperature for 1 hour. Detection was performed using the SuperSignal™ West Pico Plus kit (Thermo Scientific) and an ImageQuant LAS 500 camera (GE Healthcare). Quantification was performed on ImageJ by measuring the integral of the optical density profile of the band of the expected molecular weight. No background correction was performed.

Total proteins were extracted with RIPA lysis buffer (CWbiotech, Shanghai, China) supplemented with protease inhibitors and phosphatase inhibitors (CWbiotech, Shanghai, China). The protein concentration was measured by a BCA assay (CWbiotech, Shanghai, China). The samples were separated by electrophoresis on 8% SDS-PAGE and then transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA, United States). The membranes were blocked with PBST with 5% w/v BSA for 1 h at room temperature and then incubated with primary antibodies anti-MMP9 (10375-2-AP, Proteintech, China, 1:1,500) overnight at 4°C. The membranes were washed thoroughly with PBST. The membranes were incubated with the secondary antibody (CW0103, CWbiotech, China, 1:3,000) for 1 h at room temperature and rinsed thoroughly with PBST. Immune reactivity was detected using SuperSignal West Pico PLUS kit (Thermofisher Scientific, Waltham, MA, United States) under a Tanon 4,600 chemiluminescent imaging system (Tanon, Shanghai, China). Relative protein levels were calculated after normalization to GAPDH, which was used as a loading control.

Receptor expression was measured by ELISA in parallel for each experiment in a poly-D-lysine coated, white-walled, clear-bottom 96-well plate. Cells were fixed with 4% paraformaldehyde (EMS, ref: 15710) in PBS for 15 min at RT and blocked with 2% BSA for 45 min. After that, 45 min incubations, first with an anti-HA antibody (Thermofisher, ref: 26183) at a dilution of 1:500 followed by a second anti-mouse IgG antibody (CST, ref: 7076S) at 1:2000 dilution, were performed. Finally, chemiluminescence was recorded at the FlexStation3 after 10 min incubation with substrates A and B of SuperSignal West Pico PLUS kit (Thermofisher, ref: 34577). Average values from three replicates were normalized to WT HA₃-CXCR4 expression.