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. 2022 Sep 13;298(10):102484. doi: 10.1016/j.jbc.2022.102484

Figure 3.

Figure 3

IP/MS of STUB1 and CHIC2 reveals that STUB1 and CHIC2 interact, and further analysis shows that the TPR domain of STUB1 and C terminus of CHIC2 are required for the interaction.A, volcano plot of STUB1-V5 IP comparing STUB1 IP in Chic2 WT 32D cells to Luciferase control IP from n = 4 replicates. Labeled datapoints have p-value < 0.01, and CHIC2 is highlighted in red. B, volcano plot of CHIC2-V5 IP comparing CHIC2 IP in Stub1 WT 32D cells to Luciferase control IP from n = 4 replicates. Labeled datapoints have p-value < 0.1, and STUB1 is highlighted in red. C, Western blots of anti-V5 IP of V5-CHIC2 and whole cell lysates for V5, Stub1, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. D, volcano plot of STUB1 IP in Chic2 WT 32D cells compared to STUB1 IP in Chic2 KO 32D cells from n = 4 replicates. Labeled datapoints are those with p-value < 0.01 from STUB1 IP in Chic2 WT cells compared to Luciferase control (Fig. 4B), and CHIC2 is highlighted in red. E, volcano plot of CHIC2 IP in Stub1 WT 32D cells compared to CHIC2 IP in Stub1 KO 32D cells from n = 4 replicates. Labeled datapoints are those with p-value < 0.1 from CHIC2 IP in Stub1 WT cells compared to Luciferase control (Fig. 4C), and STUB1 is highlighted in red. F, diagram of known CHIC2 and STUB1 protein domains. G, amino acid sequences of the C-terminal 15 amino acids of CHIC family members (CHIC1, CHIC2), HSP70 family members (HS71A), and HSP90 family members (HS90A). H, Western blots of anti-V5 IP of V5-CHIC2 or V5-CHIC2 ΔIFRPD and whole cell lysate for V5, Stub1, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. I, Western blots of anti-V5 IP of STUB1-V5 or STUB1 ΔTPR-V5 and whole cell lysate for V5, Chic2, and Vinculin from 32D Cas9 cells cultured in 0.01 ng/ml IL-3. IP, immunoprecipitation; MS, mass spectrometry.