Fig. 7: RAD23B is required for SIPAN formation.

a Levels of ubiquitin and conjugated ubiquitin following nutrient deprivation in IMR90 cells. Cells were incubated in HBSS solution and harvested at different time points for immunoblotting with anti-ubiquitin or anti-conjugated ubiquitin FK2 antibodies (representative from 2 independent experiments). b, c Ubiquitin and K48-conjugated ubiquitin co-localize with SIPAN. Immunostaining of PSMD11 or PSMD4 and ubiquitin or K48 ubiquitin chains following nutrient starvation. IMR90 cells were incubated in HBSS for 6 hrs and harvested for immunostaining as indicated (representative from 3 independent experiments). d MG132 treatment increase SIPAN intensity and promote their formation. IMR90 cells were incubated in HBSS for 3 hrs in the presence or absence of MG132, and cells were harvested for immunostaining (representative from 3 independent experiments). Foci per cell (e) and foci intensity (f) were measured and represented by violin plot (representative from 3 independent experiments). g Schematic representation of treatment experiments with TAK-243, MG132, Bortezomib (BTZ) and b-AP15 inhibitors. h E1 ubiquitin-activating enzyme is required for SIPAN formation. Cells were incubated with TAK-243 E1 inhibitor for 6 hrs in HBSS (n = 3 independent experiments). i Continuous ubiquitination is required for SIPAN stability. Pre-formed SIPAN were treated with TAK-243 in HBSS for 1 hr (n = 3 independent experiments). j Ubiquitination is not required for SIPAN resolution. IMR90 cells were treated with TAK-243 for 1 hr in complete media (n = 3 independent experiments). k Deubiquitination is required for SIPAN resolution. Cells were incubated in HBSS solution for 6 hrs and then treated with b-AP15 deubiquitinase inhibitor, MG132, or BTZ in normal culture medium for 1 hr (n = 3 independent experiments). l RAD23B and other ubiquitin receptors are required for SIPAN formation. Following siRNA depletion of several ubiquitin-binding proteins and shuttling factors, IMR90 cells were incubated in HBSS for 6 hrs and harvested for immunostaining for SIPAN formation (n = 3 independent experiments). m Validation of RAD23B using additional siRNAs. Following siRNA transfection, IMR90 cells were incubated in HBSS for 6 hrs and harvested for immunostaining for SIPAN formation (representative from 3 independent experiments). n RAD23B is assembled in SIPAN following nutrient starvation. IMR90 cells were incubated in HBSS for 6 hrs and harvested for immunostaining for endogenous RAD23B and PSMD4 (representative from 3 independent experiments). o, p In yeast, RAD23 is important for PSGs formation under conditions of carbon depletion (n = 3 independent experiments). Data in graphs h, i, j, k, l, p represent the mean ± SD. 2-sided unpaired Student’s t-test (h, i, l, p). Data in e, f represent the median with interquartile range for one representative experiment. 2-sided Mann–Whitney test (e, f). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. Source data are provided as a Source data file.