Figure S5. Non-arginylatable tubulin... | Rockefeller University Press

Figure S5.

$Non-arginylatable tubulin constructs Flag-TUBA1BE77Aand TUBB5D74A-Flag can be incorporated into the microtubule cytoskeleton similarly to wild-type tubulin. (A) Sanger sequencing confirmation of Flag-TUBA1BWT, Flag-TUBA1BE77A, TUBB5WT-Flag, and TUBB5D74A-Flag. (B) Immunofluorescence of wild-type MEFs overexpressing Flag-TUBA1BWT, Flag-TUBA1BE77A, TUBB5WT-Flag, or TUBB5D74A-Flag, showing incorporation of the Flag-tagged tubulin constructs (magenta) into the normal microtubule cytoskeleton (green). Scale bar = 10 μm. (C) Western blots for Flag (top, red) and actin (bottom, green) on cell lysates from wild-type MEFs overexpressing Flag-TUBA1BWT, Flag-TUBA1BE77A, TUBB5WT-Flag, or TUBB5D74A-Flag and treated with 100 μg/ml CHX for 0–6 h. (D) Quantification of the Flag to actin (loading control) signal ratio normalized to t = 0. Mean ± SD, N = 4, two-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. (E) Western blots for Flag (top, green) and tubulin (bottom, green) on soluble and polymerized tubulin fractions from cells overexpressing Flag-TUBA1BWT, Flag-TUBA1BE77A, TUBB5WT-Flag, or TUBB5D74A-Flag. Note that the tubulin loading control images (bottom) are the same as the ones in the main text Fig. 6 A, since the two blots shown come from the same experimental repeat. (F) Quantification of the fraction of Flag-tubulin (top) or total tubulin (bottom) in the polymerized tubulin fraction. Mean ± SD, N = 4, paired t test. Symbol colors indicate paired average measurements. Source data are available for this figure: SourceData FS5. Refer to the image caption for details.$

Non-arginylatable tubulin constructs Flag-TUBA1B ^E77A and TUBB5 ^D74A -Flag can be incorporated into the microtubule cytoskeleton similarly to wild-type tubulin. (A) Sanger sequencing confirmation of Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, and TUBB5^D74A-Flag. (B) Immunofluorescence of wild-type MEFs overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag, showing incorporation of the Flag-tagged tubulin constructs (magenta) into the normal microtubule cytoskeleton (green). Scale bar = 10 μm. (C) Western blots for Flag (top, red) and actin (bottom, green) on cell lysates from wild-type MEFs overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag and treated with 100 μg/ml CHX for 0–6 h. (D) Quantification of the Flag to actin (loading control) signal ratio normalized to t = 0. Mean ± SD, N = 4, two-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. (E) Western blots for Flag (top, green) and tubulin (bottom, green) on soluble and polymerized tubulin fractions from cells overexpressing Flag-TUBA1B^WT, Flag-TUBA1B^E77A, TUBB5^WT-Flag, or TUBB5^D74A-Flag. Note that the tubulin loading control images (bottom) are the same as the ones in the main text Fig. 6 A, since the two blots shown come from the same experimental repeat. (F) Quantification of the fraction of Flag-tubulin (top) or total tubulin (bottom) in the polymerized tubulin fraction. Mean ± SD, N = 4, paired t test. Symbol colors indicate paired average measurements. Source data are available for this figure: SourceData FS5.