Figure 1.
Recurrent RPS15 mutations alter protein stability. (A) MEC-1 and HEK293T cell lines stably expressing the indicated GFP-fusion constructs were subjected to western blot analysis with anti-GFP (for GFP and GFP-RPS15 detection), RPS15, RPL11, and β-actin. Note that RPL11 detection was carried out with the same samples run in parallel on an identical blot. (B) Relative DNA levels of transduced GFP-RPS15 (wild-type and mutants) lentiviral vectors in MEC-1 and HEK293T cells determined by qPCR analysis. ACTB was used as normalization control of endogenous genomic DNA. Data are shown as the mean of 3 independent experiments + standard error of the mean (SEM). (C) Real-time qPCR against GFP-fused RPS15 constructs in the same cell lines as in (B). β-Actin mRNA levels were used as normalization controls. Data are shown as the mean of ≥3 independent determinations + SEM. (D) HEK293T cells stably expressing the GFP-RPS15 forms were treated for the indicated times with 100 mg/mL cycloheximide (CHX) and 100 nM actinomycin D (ACTD). GFP, GFP-RPS15, and β-actin (as a loading control) were detected by immunoblotting. A representative western blot of 2 independent experiments is shown. (E) HEK293T cells stably expressing the GFP-RPS15 forms were treated or not with 100 nM bortezomib (Bz) for 24 hours and subjected to western blot analysis with antibodies against polyubiquitin (PolyUb), RPS15, GFP, and β-actin as a loading control. A representative western blot of 3 independent experiments is shown. (F) Wild-type and mutant GFP-RPS15 forms were transiently coexpressed with 4xHA-tagged ubiquitin in HEK293T cells for 24 hours and incubated in the absence or presence of 100 nM Bz for an additional 12 hours. GFP-tagged RPS15 forms were immunoprecipitated with anti-GFP antibody. Then, hemagglutinin (HA; to detect polyubiquitination), GFP, GFP-RPS15, and RPL11 (as positive control of coimmunoprecipitation) were detected by immunoblotting. (G) Western blot analysis with anti-RPS15, RPL11, and β-actin of a cohort of 4 CLL-RPS15WT and 4 CLL-RPS15MUT patient samples, using MEC-1 cells as control.

Recurrent RPS15 mutations alter protein stability. (A) MEC-1 and HEK293T cell lines stably expressing the indicated GFP-fusion constructs were subjected to western blot analysis with anti-GFP (for GFP and GFP-RPS15 detection), RPS15, RPL11, and β-actin. Note that RPL11 detection was carried out with the same samples run in parallel on an identical blot. (B) Relative DNA levels of transduced GFP-RPS15 (wild-type and mutants) lentiviral vectors in MEC-1 and HEK293T cells determined by qPCR analysis. ACTB was used as normalization control of endogenous genomic DNA. Data are shown as the mean of 3 independent experiments + standard error of the mean (SEM). (C) Real-time qPCR against GFP-fused RPS15 constructs in the same cell lines as in (B). β-Actin mRNA levels were used as normalization controls. Data are shown as the mean of ≥3 independent determinations + SEM. (D) HEK293T cells stably expressing the GFP-RPS15 forms were treated for the indicated times with 100 mg/mL cycloheximide (CHX) and 100 nM actinomycin D (ACTD). GFP, GFP-RPS15, and β-actin (as a loading control) were detected by immunoblotting. A representative western blot of 2 independent experiments is shown. (E) HEK293T cells stably expressing the GFP-RPS15 forms were treated or not with 100 nM bortezomib (Bz) for 24 hours and subjected to western blot analysis with antibodies against polyubiquitin (PolyUb), RPS15, GFP, and β-actin as a loading control. A representative western blot of 3 independent experiments is shown. (F) Wild-type and mutant GFP-RPS15 forms were transiently coexpressed with 4xHA-tagged ubiquitin in HEK293T cells for 24 hours and incubated in the absence or presence of 100 nM Bz for an additional 12 hours. GFP-tagged RPS15 forms were immunoprecipitated with anti-GFP antibody. Then, hemagglutinin (HA; to detect polyubiquitination), GFP, GFP-RPS15, and RPL11 (as positive control of coimmunoprecipitation) were detected by immunoblotting. (G) Western blot analysis with anti-RPS15, RPL11, and β-actin of a cohort of 4 CLL-RPS15WT and 4 CLL-RPS15MUT patient samples, using MEC-1 cells as control.

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