full western blot images  (Mendeley Ltd)

Journal: Cell reports

Article Title: 19S proteasome loss regulates mitotic spindle assembly through a ubiquitin-independent degradation mechanism

doi: 10.1016/j.celrep.2025.116041

Figure Lengend Snippet: (A) Volcano plot comparing protein abundance as measured by quantitative MS in cells treated with 50 nM PSMD1 siRNAs and cells treated with 50 nM control siRNAs. PSMD1 and KIF11 are highlighted in red. (B) Representative immunofluorescence images of KIF11-inducible knockout cells and cells treated with 10 μM S -trityl-L-cysteine (STLC). Scale bars: 20 μm. Cells were fixed and stained on different days, and brightness is not scaled identically. (C) Western blot of cells treated with control siRNAs, cells treated with PSMD1 siRNAs, and inducible KIF11 knockout cells. The blot was incubated with a KIF11 antibody. (D) Western blots of a control or GFP-KIF11-expressing cell line treated with 50 nM control (−) or PSMD1 (+) siRNAs. The blots were incubated with KIF11, GFP, and PSMD1 antibodies. Separate blots were used for each antibody with the same samples and same amounts loaded. (E) Representative immunofluorescent images of monopolar mitotic cells showing KIF11 levels under different conditions: SAS6-inducible knockout cells (positive control), control cells treated with 10 μM STLC, KIF11-inducible knockout cells, and PSMD1 inducible knockout cells. Scale bar: 5 μm. (F) Quantification of KIF11 fluorescence intensity in monopolar mitotic cells under different conditions. Fluorescence intensity values were normalized to levels in PSMD1 knockout cells. Bars represent mean ± standard deviation. p values were calculated with two-tailed Welch’s t tests comparing each condition to PSMD1 knockout: p = 0.0275 for STLC, p = 0.0173 for SAS6 knockout, and p < 0.0001 for KIF11 knockout. * p < 0.05, **** p < 0.0001. The experiment was replicated 4 times; 29–92 cells were quantified for each condition for each replicate. (G) Western blots of cells treated with either 50 nM control siRNAs, 50 nM PSMB7 siRNAs, 50 nM PSMD1 siRNAs, or 10 μM MG132. Blots were incubated with a KIF11 antibody, PSMD1 antibody, and PSMB7 antibody. Separate blots were used for each antibody with the same samples and same amounts loaded. (H) Western blot of cells transduced with a lentivirus containing the mCherry-expressing gene knockout plasmids for PSMD1, PSMD8, PSMD11, PSMA6, or PSMC4. Cells were sorted for mCherry. Control cells are untransduced parental cells. The blot was incubated with a KIF11 antibody.

Article Snippet: Full Western Blot Images , Mendeley Data , Mendeley Data: https://doi.org/10.17632/k49vv5dh9p.1.

Techniques: Quantitative Proteomics, Control, Immunofluorescence, Knock-Out, Staining, Western Blot, Incubation, Expressing, Positive Control, Fluorescence, Standard Deviation, Two Tailed Test, Transduction, Gene Knockout

Journal: Cell reports

Article Title: 19S proteasome loss regulates mitotic spindle assembly through a ubiquitin-independent degradation mechanism

doi: 10.1016/j.celrep.2025.116041

Figure Lengend Snippet: (A) Western blots of cells treated with 50 nM siRNAs and the indicated compounds. MG-132 was used at 10 μM and TAK-243 at 300 nM. Blots were incubated with KIF11, PSMD1, and MDM2 antibodies. Separate blots were used with the same samples and same amounts loaded. (B) Western blot of cell lines expressing the indicated GFP-KIF11 truncation constructs treated with 50 nM control (−) or PSMD1 (+) siRNAs. The blot was incubated with a GFP antibody. Lower and higher exposure times are shown. (C) Western blot of cell lines expressing the indicated GFP-KIF11 truncation constructs treated with 50 nM control (−) or PSMD1 (+) siRNAs. The blot was incubated with GFP and PSMD1 antibodies. Lower and higher exposure times are shown. (D) Western blot of control cells or a cell line expressing a C-terminally tagged KIF11-GFP construct. Cells were treated with either 50 nM control (−) or PSMD1 (+) siRNAs. The blot was incubated with KIF11 and PSMD1 antibodies. (E) Quantification of the immunofluorescence experiment for cells expressing KIF11 constructs. The bar graph shows the percentage of mitotic cells with monopolar spindles for parental HeLa cells, a cell line expressing N-terminally tagged GFP-KIF11, and a cell line expressing C-terminally tagged KIF11-GFP treated with 12.5 nM PSMD1 siRNAs. p values were calculated with two-tailed Welch’s t tests; p = 0.0039 and p = 0.3703 between HeLa and KIF11-GFP and GFP-KIF11, respectively. ns = not significant, ** p < 0.01. (F) Representative immunofluorescence images from (E). Scale bar: 10 μm. (G) Bar graph showing fold change of protein abundance pulled down in mitotic compared to asynchronous cells, as measured by quantitative MS. GFP IP was performed on GFP-PSMB4 cell lines in mitotic (STLC-arrested) and asynchronous cells. The bar graph shows the abundance ratios of mitotic/asynchronous values for proteasome 19S and 20S subunits.

Article Snippet: Full Western Blot Images , Mendeley Data , Mendeley Data: https://doi.org/10.17632/k49vv5dh9p.1.

Techniques: Western Blot, Incubation, Expressing, Construct, Control, Immunofluorescence, Two Tailed Test, Quantitative Proteomics