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. Author manuscript; available in PMC: 2020 Aug 4.
Published in final edited form as: Sci Signal. 2020 Feb 4;13(617):eaav1256. doi: 10.1126/scisignal.aav1256

Fig. 5. PACRG restores defective NF-κB activation and prevents increased cell death in SHARPIN-deficient cells.

Fig. 5

(A) HEK293T cells were transfected with control, SHARPIN-, or PACRG-specific siRNAs with or without PACRG or SHARPIN plasmids as indicated, then the cells were treated with TNF and lysed under denaturing conditions. Linear ubiquitination was analyzed by UBAN affinity purification by Strep-TagII beads followed by immunoblotting for M1-linked ubiquitin. PACRG knockdown efficiency was determined by real-time RT-PCR. Blot is representative of 3 independent experiments. (B) HEK293T cells were transfected with HA-NEMO and either PACRG or SHARPIN, then lysed under denaturing conditions followed by immunoprecipitation of NEMO by anti-HA agarose. Anti-c-Myc-agarose was used as a control for nonspecific binding. Immunoprecipitated NEMO was immunoblotted for M1-linked ubiquitin and NEMO. The input was immunoblotted for NEMO, PACRG, SHARPIN, and β-actin. Blot is representative of 3 independent experiments. (C) Wild-type (WT) and cpdm MEFs were transfected with SHARPIN, PACRG or EGFP (negative control), then the cells were treated with TNF. The fraction of cells showing nuclear translocation of p65 was determined for each condition. Data represent the mean ± SEM of 3-4 independent experiments. For statistical analysis one-tailed Mann-Whitney U-test was performed. At least 100 cells were analyzed per condition. (D) Cpdm MEFs were transfected with either SHARPIN or PACRG. Transfection of EGFP was used as a control. After TNF treatment, apoptotic cell death was quantified by counting transfected cells positive for active caspase-3. Data represent the mean ± SEM of 3 independent experiments. For statistical analysis one-tailed Mann-Whitney U-test was performed. At least 100 cells were analyzed per condition. (E) MEFs were transiently transfected with the indicated siRNAs and lysed after 48 h. Lysates were analyzed by immunoblotting for HOIP, SHARPIN, and β-actin. PACRG knockdown efficiency was determined by real-time RT-PCR. Data represent the mean ± SEM of 5 independent experiments, intensity normalized to β-actin presented as ratio to total intensity of all three bands. For statistical analysis ANOVA followed by Tuckey’s Multiple Comparison Test was performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001.