Rates of protein synthesis are regulated by conditions that alter available GTP. (A and B) Quantification of protein lysates from AHA-labeled BJ-5ta cells treated with (A) MPA or cycloheximide (CHX) and (B) analyzed by SDS-PAGE. AHA signal was normalized to Coomassie stain. Results are from three independent replicates. (C and D) Quantification of protein lysates from AHA-labeled BJ-5ta cells treated with (C) leptomycin B and (D) analyzed by SDS-PAGE. AHA signal was normalized to Coomassie stain. Results are from three independent replicates. (E) Quantification of GEVAL ratiometric signal (405 nm/488 nm) in BJ-5ta cells expressing GEVALNull or GEVAL30 and treated with leptomycin B (GEVALNull control n = 183, LeptoB n = 146; GEVAL30 control n = 156, LeptoB n = 135). Results are from three independent replicates. (F) Quantification of protein synthesis and representative images of BJ-5ta cells plated on increasing substrate rigidities, then fixed and labeled with AHA (1 kPa n = 646, 22 kPa n = 833, 46 kPa n = 793, 308 kPa n = 530). Results are from three independent replicates. Scale bar 10 µm. (G and H) Quantification of protein lysates from AHA-labeled BJ-5ta cells depleted of (G) SUN2 and (H) analyzed by SDS-PAGE. AHA signal was normalized to Coomassie stain. Results are from four independent replicates. Significance was calculated using one-way ANOVA with Dunnett’s post hoc (A), unpaired t test (C, E, and G), or one-way ANOVA with Tukey’s post hoc (F). ns P > 0.05, P*<0.05. P****<0.0001. Source data are available for this figure: SourceData F10.