Ikzf1

Chemicals such as lenalidomide, bortezomib, MG132, bafilomycin A1, hydroxychloroquine, PD150606, Calpeptin, BAPTA, Ionomycin, E64, pepstatinA, and zVAD.fmk were obtained from Sigma and were used in this study. Antibodies against Actin, p62, IKZF1, BIM, Bcl2, cIAP2, and XIAP1 (Santa Cruz Biotechnology), LC3, Caspase-3, and PARP (Cell Signaling Technology), IRF4 and IKZF3 (Abcam), CD38 FITC conjugate (BD Biosciences), and anti-mouse and anti-rabbit secondary antibodies conjugated with horseradish peroxidase (Cell Signaling Technology), and with Alexa Fluor 594 (Invitrogen) were also used for Western blotting, immunofluorescence, and flow cytometry assays.

This was done as previously reported by us (13 (link)). In summary, the myeloma cells were treated with drugs for 24 hours and cytospun slides were made. The cells were fixed in 4% paraformaldehyde followed by blocking using 5% goat serum. It was further incubated with primary antibodies such as IKZF1 (Santa Cruz Biotechnology) overnight at 4°C. The slides were rinsed with PBS thrice and incubated with secondary antibodies (anti-mouse) conjugated with Alexa Fluor 594, (Invitrogen) for 1 hour. The slides were again washed, air dried, and counterstained with DAPI containing mountant (Vectashield). The images were acquired in fluorescence microscope (Axioimager M1, Carl Zeiss) at 100 × with oil immersion and images were analyzed using ISIS Metasystem (Metasystems GmbH).

Total protein lysates were obtained from cells by lysing the samples in cold RIPA buffer in the presence of phenylmethylsulfonyl fluoride (PMSF), sodium orthovanadate, and protease and phosphatase inhibitors (all from ThermoFisher Scientific). Protein concentration was evaluated by a BCA assay (23225; Pierce). Western blot analysis was performed with antibodies to the following proteins: GPR68 (sc-79622; Santa Cruz), CRBN (SAB2106014; Sigma), CAPN1 (2556; Cell Signaling Technology), CAPN2 (2539; Cell Signaling Technology), CAST (4146; Cell Signaling Technology), IKZF1 (sc-398265; Santa Cruz), CK1-α (sc-6477; Santa Cruz), and GAPDH (5174; Cell Signaling Technology). Anti-IKZF1 (1:500), anti-GPR68 (1:800), and all other primary antibodies (1:1,000) were diluted in 1× TBST buffer (Cell Signaling) containing 5% BSA or 5% milk. All secondary antibodies are conjugated with horseradish peroxidase (HRP). Goat anti-rabbit (111-035-003; Jackson ImmunoResearch) and goat anti-mouse (115-035-003; Jackson ImmunoResearch) secondary antibodies were diluted at 1:10,000 in 1× TBST buffer containing 5% milk. Donkey anti-goat (sc-2020; Santa Cruz) antibody was diluted at 1:3,000. ECL Western Blotting Substrate (32106; Pierce) and Supersignal West Femto (34095; Pierce Chemical) were used for detection of HRP on immunoblots with X-ray film or with a ChemiDoc Touch Imaging System (Bio-Rad).