H3663

MEF cells stably expressing GFP-Rab10 were transiently transfected with plasmids carrying wild-type HA-PPM1H, HA-PPM1H[H153D], or HA-PPM1H[D288A]. After 24 h the cells were fixed with 4% (by vol) paraformaldehyde for 10 min, permeabilized with 0.1% saponin for 15 min, and blocked with 1% (by mass) BSA for 1 h. Cells were subsequently stained with mouse anti-HA antibody 1:1000 (Sigma-Aldrich H3663) and rabbit phospho-Rab10 1:1000 (Abcam ab230261). Highly cross absorbed H+L secondary antibodies (Life Technologies) conjugated to Alexa 568 or Alexa 647 were used at 1:5000. Primary and secondary antibody incubations were for 1 h at room temperature. All images were obtained using a spinning disk confocal microscope (Yokogawa) with an electron multiplying charge coupled device (EMCCD) camera (Andor, UK) and a 20x1.4NA or 40x1.4NA objective. Images were analyzed using CellProfiler and presented as maximum intensity projections. Results were quantified by determining the ratios of phospho-Rab10 signal to GFP-Rab10 in cells expressing wild-type HA-PPM1H, HA-PPM1H[H153D], or HA-PPM1H[D288A], and normalizing these numbers to the phospho-Rab10/GFP-Rab10 ratio in non-expressing cells. For each condition at least 20 cells were analyzed. Significance was determined by one-way analysis of variance with Dunnett's post-test at 95% confidence interval. ***, P < 0.001.

For immunocytochemical analysis, cells were fixed in a 4% paraformaldehyde solution in PBS for 10 min at room temperature (RT), permeabilized in a 0.5% Triton-X-100 solution in PBS for 5 min and blocked for 1 h with 3% BSA in PBS. Immunostaining was performed by addition of the primary antibodies (mouse anti-HA, Sigma Aldrich, H3663, 1:1000; rabbit anti-GRP75, Abcam, ab53098; 1:5000; goat anti-c-myc, Novus Biologicals, NB600335, 1:500) in the blocking solution overnight at 4 °C and the appropriate fluorescently labelled secondary antibodies for 1 h at RT. Nuclei were stained by addition of DAPI. Images were acquired using a Leica SP8-X confocal microscope (Leica Microsystems) and analyzed using Imaris software (Oxford Instruments).