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

Figure 2.

Figure 2

Stub1 and Chic2 KO lead to increased endogenous total and cell surface Csf2rb.A, Western blots of 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 for STUB1 and Vinculin. B, Western blots of 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 for Chic2 and Vinculin. C, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3 (S.E. = short exposure, L.E. = long exposure). Representative of three independent biological replicates with similar results. D, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 cultured in 5, 0.1, or 0.01 ng/ml IL-3. Representative of three independent biological replicates with similar results. E, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgStub1-1, or sgStub1-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. F, Western blots of Csf2rb and Vinculin in 32D Cas9 cells with sgNT, sgChic2-1, or sgChic2-2 treated with 10 μM cycloheximide (CHX) for 0, 2, 4, or 8 h. G, Western blots of Csf2rb, Stub1, Chic2, and Vinculin in 32D Cas9 cells with sgNT/sgNT, sgStub1-1/sgNT, sgNT/sgChic2-1, or sgStub1-1/sgChic2-2. H, bar graph showing ratio of mean GFP to mean mCherry signal of Csf2rb reporter in 32D cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/ml IL-3 as measured by flow cytometry. Bars show GFP/mCherry ± SD from n = 3 replicates. I, bar graph showing median fluorescence intensity for anti-Csf2rb-PE (CD131) in 32D Cas9 cells with sgNT, sgChic2-1/2, or sgStub1-1/2 in 0.01 ng/m IL-3 as measured by flow cytometry. Bars show mean ± SD from n = 3 replicates. p-values calculated by unpaired Student’s t test between sgNT and other conditions at each cytokine concentration.