Spinning disk confocal microscope

Following fixation in 4% PFA overnight, half of the right kidney was cryoprotected in 30% sucrose overnight, embedded in Optimal Cutting Temperature (OCT) medium and sectioned at 10 μm. For IF staining, sections were fixed with 4% PFA for 10 min, permeabilized with 0.2% Triton X-100 for 10 min and incubated in blocking solution [1% bovine serum albumin (BSA), 2% donkey serum and 0.02% sodium azide into 1× PBS] for 30 min at room temperature. Sections were incubated in primary antibody overnight at 4°C according to the manufacturer's recommendations, washed with PBS and incubated with the appropriate secondary antibodies in blocking solution for 1 h at room temperature (primary and secondary antibodies are listed in Table S2). After the addition of secondary antibodies, nuclei were stained by Hoechst (Sigma-Aldrich), and samples were mounted using IMMU-MOUNT (Thermo Fisher Scientific). All fluorescence images were captured on a Nikon Spinning-disk confocal microscope with a Yokogawa X1 disk, using a Hamamatsu flash4 sCMOS camera. Images were processed and analyzed using NIS Elements software (Nikon) version 5.0 and ImageJ.

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.

Transient expression assays were conducted in Nicotiana benthamiana leaves to determine the subcellular localization of PvTET3 and PvTET6 proteins. The molecular construction carrying 35S:PvTET3-GFP and 35S:PvTET6-GFP was transferred to A. tumefaciens AGL1. For transient assays, leaves from 4- to 6-week-old wild-type N. benthamiana plants were coinfiltrated with the agrobacterium suspension harboring 35S:PvTETx-GFP. The infiltrated plants were marked and kept in a growth room at 16 h light/8 h darkness at 25 ± 2°C. Plasmolysis was induced using a 1M NaCl hypertonic solution. Fluorescence was visualized 48–72 h after infiltration using a spinning disk confocal microscope (Yokogawa, Japan) as described below.

Images were acquired with a spinning disk confocal microscope (Yokogawa). A healthy-appearing neuron that was about two cell diameters away from its neighbors was placed in the center of the camera field to capture digital images of fluorescence emissions at 488 nm and 594 nm using MetaMorph image capture software. The selected cell was imaged in serial optical sections at 0.33 μm intervals over a total depth of 5 μm, for a total of 15 optical sections. Maximum intensity projections (MIPs) were generated from these sections, yielding 5 MIPs representing 1 μm of depth each; fluorescence intensity in each MIP was quantified as described previously (Ippolito and Eroglu, 2010 (link)). Synaptic puncta, defined by co-localization of synaptophysin and PSD-95 labeling, were quantified in the selected regions of interest (ROI; 89 × 89 μm square) using Puncta Analyzer program written by Bary Wark for ImageJ 1.26.¹ A total of 9 cells from 3 coverslips (each coverslip from a different culture batch) were analyzed for each experimental condition.