Figure 2: MYPT1 functions as a switch to regulate contractility and microtubule acetylation. | Nature Communications

Figure 2: MYPT1 functions as a switch to regulate contractility and microtubule acetylation.

From: MYPT1 regulates contractility and microtubule acetylation to modulate integrin adhesions and matrix assembly

Figure 2

(a) Representative western blot probed with an anti-MYPT1 antibody after immunoprecipitating HDAC6 after the indicated treatments. Two percent of input (whole-cell lysate, WCL) and 50% of the immunoprecipitates were loaded. Intensities of immunoprecipitated bands were 0.82±0.07 (TSA) and 1.00±0.09 (NaB) compared with the control (1.0; n=3). (b) Representative western blot of GST pull-down of soluble recombinant HDAC6 using recombinant MYPT1 immobilized on glutathione-agarose beads at an equal molar ratio. Five percent of the input and 50% of the pull-downs were loaded and probed with the indicated antibody. (c) Western blot after immunoprecipitating MLC with TSA treatment and probed with an anti-MYPT1 antibody. Two percent of input (whole-cell lysate, WCL) and 50% of the immunoprecipitates were loaded. (d) Representative western blot after immunoprecipitating MLC in the presence of purified recombinant HDAC6. Five percent of input and 100% of immunoprecipitates were loaded and probed with an anti-MYPT1 antibody. Intensity of immunoprecipitated band was 0.87±0.05 compared with the control (1.0; n=4). (e) Representative western blot after immunoprecipitating HDAC6 with TSA treatment and probed with an anti-pSer antibody. Fifty percent of the immunoprecipitates were loaded. Intensity of immunoprecipitated band was 1.47±0.20 compared with the control (1.0; n=4). (f) Schematic summary of findings in a–e. (g) Western blot of HDAC6 immunoprecipitates from HFFs transfected with scrambled siRNA (control siRNA) or siRNA silencing MYPT1 and then probed with the indicated antibodies. Two percent of the input (WCL) and 100% of the immunoprecipitates were loaded. Intensity of MYPT1 band after transfection was 0.35±0.04 compared with the control (1.0; n=7), and immunoprecipitated band was 2.32±0.27 compared with the control (1.0; n=4). (h) Western blot of whole-cell lysates from cells transfected with scrambled siRNA (control siRNA), siRNA silencing HDAC6 or siRNA silencing MYPT1. Each lysate was probed with the indicated antibodies. Intensity of each band probed with an anti-acetylated tubulin antibody normalized against GAPDH was 1.43±0.21 (HDAC6 siRNA), 0.79±0.11 (MYPT1 siRNA) compared with the control (1.0; n=3). (i) Western blot of whole-cell lysates from cells overexpressing HDAC6 mutated at Ser-22, as indicated in each lane, and probed with the indicated antibodies. Intensity of each bands probed with an anti-acetylated tubulin antibody normalized against GAPDH were 1.04±0.09 (S22A-HDAC6-Flag), 0.82±0.12 (S22E-HDAC6-Flag) compared with the control (1.0; n>6). Data are mean±s.e.m.

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