Results: 4

1.
Figure 1

Figure 1. Arginylated γ-actin is selectively degraded. From: Differential Arginylation of Actin Isoforms is Regulated by Coding-Sequence-Dependent Degradation.

A. Top: Representative immunoblots of the lysates of HEK 293T cells transfected with actin-GFP constructs probed with anti-GFP and β-tubulin as a loading control. Boxed numbers at the bottom indicate the specific mRNA levels in each sample. Bottom: Quantifications of the stationary protein levels as the amounts of in vivo actin-GFP per mRNA unit, normalized to the level of M-β-actin (n= 4 for R-β, R-γ and 6 for M-β, M-γ). B. Changes in actin protein level over time in the presence of cycloheximide, normalized to zero time point (n= 3 for M-β, M-γ and 6 for R-β, R-γ). C. Top: representative immunoblots showing the stationary protein levels of the actin fusions and β-tubulin loading control in cells treated with DMSO or MG132. Bottom, quantification of the fold change of each fusion protein upon MG132 treatment compared to the DMSO-treated sample (n= 3 for M-β, M-γ, R-β and 6 for R-γ). Numbers in all panels represent mean +/-SEM; ** P<0.01, Student's t-test.

Fangliang Zhang, et al. Science. ;329(5998):1534-1537.
2.
Figure 4

Figure 4. Co-translational degradation of arginylated γ-actin is achieved via a ubiquitin-dependent mechanism. From: Differential Arginylation of Actin Isoforms is Regulated by Coding-Sequence-Dependent Degradation.

A. Left: Representative immunoblots of the lysates of HEK 293T cells transfected with actin-GFP constructs probed with anti-GFP and β-tubulin as a loading control; specific mRNA levels are indicated below each lane. Middle: stationary protein levels generated with each construct quantified per mRNA unit and normalized to the level of arginylated γ-actin (n=4). Right: changes in actin protein levels over time in the presence of cycloheximide, normalized to the protein level at zero time point (n= 4 for K18L-Rγ and 6 for R-γ). B. Left: Total actin-GFP levels in cells co-transfected with actin constructs and His-tagged ubiquitin (input), and in the pellet after His-tag pulldown to enrich for ubiquitinated proteins (pulldown). Right: quantification of the ubiquitination levels (n=3). Levels of K18L- R-γ mutant are normalized to those of R-γ. C. Left: representative immunoblots of the input and His-tag-Ub pulldown for the constructs marked on the top. Right: quantification of the ubiquitination levels, normalized to those of R-β (n=3). Numbers in all panels represent mean +/- SEM; * P<0.05, ** P<0.01, Student's t-test.

Fangliang Zhang, et al. Science. ;329(5998):1534-1537.
3.
Figure 2

Figure 2. Coding sequence affects stationary protein levels of arginylated actin isoforms without affecting their posttranslational degradation dynamics. From: Differential Arginylation of Actin Isoforms is Regulated by Coding-Sequence-Dependent Degradation.

A, B. Representative immunoblots of the lysates of HEK 293T cells transfected with actin-GFP constructs probed with anti-GFP and β-tubulin as a loading control. Boxed numbers at the bottom indicate corresponding mRNA levels. Histogram: stationary protein levels of each actin species quantified per mRNA unit and normalized to the level of R-β- (A) or M-β-actin (B). In A, n=5; in B, n=3 for p-β and p-γ-, the constructs that contain no N-terminal Ub fusion (Fig. S2, SOM Text 2), M- γc-β, and M- βc-γ and 6 for M- β, M-γ. C. Changes in actin protein levels over time in the presence of cycloheximide, normalized to the protein level at zero time point (n= 5 for R- γc-β, R-βc- γ- and 6 for R-β, R- γ). Numbers in all panels represent mean +/- SEM; ** P<0.01, Student's t-test.

Fangliang Zhang, et al. Science. ;329(5998):1534-1537.
4.
Figure 3

Figure 3. Slower translation of γ-actin is linked to arginylation-mediated degradation. From: Differential Arginylation of Actin Isoforms is Regulated by Coding-Sequence-Dependent Degradation.

A. Representative immunoblots of the lysates of HEK 293T cells transfected with actin-GFP constructs and treated with MG132, probed with anti-GFP and β-tubulin as a loading control. B. Quantification of protein levels for M-β and M-γ-actin adjusted to the starting mRNA level and normalized to the 1 hr time point in each set (n=8). C. Incorporation of 3H-Leu into ribosome-bound nascent peptides of β- and γ-actin over time, normalized to 12′ time point (n=3). D. Pattern of 35S-labeled nascent peptides. Boxed area represents the bottom part of the same gel, contrasted to emphasize the lower molecular weight bands using Adobe Photoshop ‘brightness/contrast’ function applied equally to the entire inset image. Arrow in both images indicates the additional low molecular weight band present in γ-actin. Similar results were observed in two independent repeats. E. Stationary protein levels of actin proteins plotted against the increasing concentrations of anisomycin. F. Stationary levels of actin proteins from the experiment shown in E, quantified by independently performed Western blots by loading M-β and R-β side by side and plotted as ratio of [R-β]/[M-β] against anisomycin concentration. Curves in E and F are normalized to the sample with 0 μM of anisomycin in each set (n=3). Numbers in all panels represent mean +/- SEM.

Fangliang Zhang, et al. Science. ;329(5998):1534-1537.

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