Anti sharpin

anti-FLAG M2 (Sigma, F1804), anti-HA.11 (BioLegend, 901501), anti-myc (Santa Cruz Biotechnology, sc-40), anti-GST (Yeasen, 30902ES60), anti-α-tubulin (Yeasen, 30304ES60), anti-β-actin (Yeasen, 30101ES60), anti-GAPDH (Immunoway, YM3029), anti-CD31 (Abcam, ab28364), anti-VEGF (Novus Biologicals, NB100-664), anti-PCNA (Servicebrio, GB11010), anti-ubiquitin (Santa Cruz Biotechnology, sc-8017), anti-GFP (Yeasen, 31002ES60), anti-LUB9 (Lifesensors, AB130), anti-HIF1α (Novus, NB100-479; Abcam, ab228649), anti-HIF1β (Cell Signaling technology, #5537), anti-Otulin (Abcam, ab211328), anti-HOIP (Abcam, ab46322; R&D systems, MAB8039), anti-HOIL-1L (Millipore, MABC576), anti-Sharpin (Proteintech, 14626-1-AP), anti-LAMP2 (Santa Cruz Biotechnology, sc-18822), anti-Lamin B1 (Zenbio, R24825), normal mouse immunoglobulin (IgG) 1 (Santa Cruz Biotechnology, sc-3877), anti-mouse IgG(H + L) (Jackson ImmunoResearch, 151383), and anti-rabbit IgG(H + L) (Jackson ImmunoResearch, 145472).

We seeded 5 × 10⁵ WT (H-2^b) MEF cells (85 (link)) or B16F1 melanoma cells (provided by Miriam Merad, Icahn School of Medicine at Mount Sinai, New York, USA) into each well of a 6-well plate in complete media. We replaced the medium with 1 mL of Opti-MEM. After complexing 2 ng of control or Sharpin-targeting plasmid with 10 μL of Lipofectamine2000 in 300 μL of Opti-MEM for 5 minutes, we dispensed the solution dropwise to the designated well of the 6-well plate. We replaced the culture medium the next day. Two days after transfection, we replaced the medium again with 2 mL of complete media containing 5 μg/mL of puromycin plus plasmid PX459 V2.0 encoding a control nontargeting sgRNA (5′-GCGAGGTATTCGGCTCCGCG-3′) or targeting Sharpin (5′-CGTGCACTTCCTCGACCCCG-3′) (Genscript). We confirmed absence of SHARPIN by immunoblotting with anti-SHARPIN (Proteintech, catalog 14626-1-AP), and bulk puromycin-resistant cells were used in experiments to minimize founder effects.