Ab49763

20 µg of whole cell extracts were separated on SDS-PAGE and transferred on a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad, ON, Canada). For detection of FLAG-tagged AC isoforms, after transferring, the membranes were first blocked by incubation with 3% fish gelatin prepared TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, and the membrane was incubated with antibodies against anti-FLAG (1:1000), β-actin (1:10,000) at 4 °C for 12 h. Membranes were washed thrice with TBST for 10 min and for β-actin (Invitrogen, ON, Canada), incubated with a 1:5000 dilution of horseradish peroxidase-conjugated anti-mouse (Bio-Rad) for 2 h. Anti-FLAG was HRP conjugated (ab49763, Abcam, Cambridge, MA, USA). Blots were washed with TBST three times and developed with the clarity max ECL system (Bio-Rad) according to the manufacturer’s instructions.

The proteome was analyzed by SDS‒PAGE and transferred to PVDF membranes (Bio-Rad; BR162-0177). The membranes were blocked with 2% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h. To detect the desired proteins, the membranes were incubated overnight at 4 °C with the primary antibodies—1:1000 dilution of α-tubulin (CST; #3873), β-tubulin (CST; #2146), IκB (CST; #4814), caspase-1 (CST; #24232), cleaved caspase-1 (CST; #89332), IL-1β (CST; #31202), cleaved IL-1β (CST; #63124), acetylated α-tubulin (CST; #5335), p65 (Abcam; ab16502), FLAG (Abcam; ab49763); and 1:2,000 dilution of GAPDH (CST; #2118), β-actin (CST; #4970), and LMNB1 (Abcam; ab16048). After washing with TBST, the resulting membranes were exposed to HRP-conjugated secondary antibody—1:5000 dilution of anti-rabbit (CST; #7074) and anti-mouse (CST; #7076)—for 1 h at room temperature. After washing with TBST, the membranes were incubated with an enhanced chemiluminescence (ECL) prime kit (Cytiva; RPN2232). Chemiluminescent signals from the desired proteins were detected using ChemiDoc.

The total protein concentration was determined using the BCA protein assay kit. The protein sample (40 μg) was added to SDS-PAGE and electrophoresed at 50 V for 3 hours, and then transferred to PVDF membrane (Millipore, USA). After blocking with TBST containing 5% (w/v) nonfat dry milk for 1 hour at room temperature, anti-FLAG (Abcam, USA, #ab49763, diluted 1:2000) or mouse anti-GAPDH (Abcam, USA, #ab8245, dilution 1:2000) was incubated overnight or 4h. After washing with TBST, the membrane was incubated with HRP (horseradish peroxidase)-labelled goat anti-mouse IgG (Abcam, USA, #ab6789, dilution 1:2000) secondary antibody at room temperature for 2h. Membranes were analyzed using Ultra ECL detection reagent (Shanghai Yisen Biotechnology Co., Ltd., China).

Proteins extracted by the M-PER Mammalian Protein Extraction Reagent (Cell Signaling Technology, Inc., Danvers, MA, USA) were resolved on 10% SDS-PAGE gels (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and transferred to polyvinylidene fluoride membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). The monoclonal mouse anti-Flag (the Ad-HCV core was tagged with 3X Flag) primary antibody (1:500; ab49763; Abcam, Cambridge, UK) was used overnight at 4°C and the horseradish peroxidase-linked rabbit anti-mouse IgG (1:10,000; ab97046; Abcam) was used at room temperature for 1 h as the secondary antibody. The monoclonal mouse GAPDH antibody (1:1,000; ab8245; Abcam) was used overnight at 4°C as a loading control. Blots were developed using Supersignal WestPico chemiluminescent substrate (Pierce Biotechnology, Inc.), imaged and analyzed using the Bio-Rad ChemiDoc XRS Gel Imaging System (Bio-Rad Laboratories, Inc.).

Cells carrying the ADH5-expressing plasmid pLox-ADH5-FLAG-CT-BSR^{3 (link)} were selected using 4 µg mL⁻¹ Blasticidin (BSR). BSR-resistant cells were clonally diluted and ADH5-expression verified by western blot against FLAG epitope (Abcam, #ab49763, 1:2000).
The human GCLM gene was amplified from the MGC clone MHS6278-202809187 and cloned into the SalI-opened pAAVS1^{57 (link)} vector fused to mCherry by Gibson assembly. The primers used for GCLM amplification were 5′-TTTTGGCAAAGAATGGTCGACCGCTGCCATGGGCACCGAC-3′ and 5′-GCCCTTGCTCACCATGTCGATAGAACCCCTTCTTTTAGCTTGTAAAATG-3′. The vector generated (pAAVS1-PC-GCLM::Cherry) was confirmed by sequencing and co-transfected into HCT116 cells together with the AAVS1-TALEN-R/AAVS1-TALEN-L plasmids (Addgene #59026 and #59025, respectively). Puromycin-resistant cells were selected using 5 µg mL⁻¹ puromycin. The population expressing GCLM::Cherry was enriched using a Becton Dickinson FACSAria II cell sorter and used for complementation experiments.