Trans blot turbo semi dry transfer system

Frozen cell pellets were lysed in 1% SDS, 50 mM Tris, pH 8.0 buffer with added Halt Protease and Phosphatase Inhibitors (ThermoFisher, 10137963), and proteins were quantified by Pierce BCA Protein Assay Kit (ThermoFisher, 23227) according to the manufacturer’s protocol. Protein lysates were separated on 4–20% Mini-PROTEAN TGX Gels (Bio-Rad, 456–1096) and transferred onto polyvinylidene difluoride membrane (Bio-Rad, 170–4156) using semidry Trans-Blot Turbo Transfer System (Bio-Rad) and a high-molecular-weight standard protocol. Membranes were blocked in 5% TBS-T milk at room temperature for a minimum of 1 h with gentle rocking. Blots were incubated with primary antibodies at room temperature overnight with gentle rocking. Blots were washed three times with TBS-T and incubated with secondary antibodies for 1 h at room temperature. The antibodies used are listed in Table 3. After washing three times in TBS-T, blots were imaged using Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500).

C. reinhardtii cells were harvested 5th day after the treatment for Western blot analysis. Approximately 1.5 × 10⁶ cells of C. reinhardtii were harvested by centrifugation at 4000 rpm for 10 minutes and washed with PBS. The cell number was quantified with a hematocytometer (INCYTO, Korea) under a microscope (Nikon TS100, Tokyo, Japan). The cells were resuspended in a 100 μL mixture of Laemmli lysis buffer, beta mercaptoethanol, protease inhibitor and phosphatase inhibitor (19:1:1:1, v/v/v/v). The mixture was then heated at 100 °C for 30 min. for protein extraction. The supernatant was recovered by centrifuging the cells at 13000 RPM for 5 minutes. The protein lysate was run on a SDS page gel, which was subsequently electroblotted to a PVDF (polyvinylidene fluoride) membrane with the trans-blot turbo semi-dry transfer system (Bio-Rad). Immunodetection for the activation of ERK1/2 and MEK1/2 was done with phospho-p44/42 MAPK (Thr202/Tyr204) (Cell Signaling Technology, Beverly, MA, USA)^{49 (link)} and phospho-MEK1/2 (Ser217/221)^{50 (link)} antibodies. JNK was detected with the phospho-SAPK/JNK antibody (Cell Signaling Technology, Beverly, MA, USA)^{51 (link)}. β-subunit of ATP synthase (ATPβ) antibody was used as an experimental loading control^{52 (link)}. Signals were detected by using exposure in ChemiDoc system (Bio-Rad) with enhanced chemiluminescence substrate (Bio-Rad, USA).

Following infection, host cell supernatants were collected, precipitated using 10% v/v TCA, and washed with ice-cold acetone twice before resuspension in 2× Laemmli buffer (from 5× stock, 312.5 mm Tris-HCl, pH 6.8, 10% w/v SDS, 50% v/v glycerol, 0.5 m DTT, 0.05% w/v bromphenol blue). Infected cells were lysed with 1× Laemmli buffer and cell lysates collected on ice. All samples were boiled for 5 min prior to SDS-PAGE analysis and Western blotting. Proteins were transferred to Hybond-P PVDF membranes (GE Healthcare) using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). Membranes were blocked with 5% w/v skimmed milk in PBS with 0.1% v/v Tween 20 (PBS-T) prior to addition of primary antibodies, rabbit anti-CD55, 1:1000 (Thermo Fisher Scientific) and mouse anti-α-tubulin, 1:2000 (Sigma). HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) were used when necessary (goat anti-rabbit-HRP, 1:5000 and goat anti-mouse-HRP, 1:2000). Membranes were washed three times 5 min with PBS-T prior to development with EZ-ECL chemiluminescence reagent (Geneflow). Blots were visualized using a LAS-3000 Imager (Fujifilm). For re-blotting, blots were stripped using stripping buffer (15 mg/ml glycine, 0.5% w/v SDS, 1.0% v/v Tween 20, pH 2.2).

After SDS-PAGE of the collected SEC fractions (10 μL per fraction) on a 4–15% acrylamide gradient gel, the proteins were transferred onto a PVDF membrane using a Trans-Blot Turbo semi-dry transfer system (Bio-Rad). In the cases where the cargo proteins were fusion constructs with N- or C-terminal parts of TccC3HVR, a custom-made anti-TccC3HVR rabbit polyclonal antibody (Cambridge Research Biochemicals) was used as the primary antibody at 1:1000 dilution. For Cdc42 without a TccC3HVR fusion, an anti-Cdc42 rabbit polyclonal antibody (Cell Signaling Technology, Cat. No. 2462) was used as the primary antibody at 1:1000 dilution. For TEV, an anti-TEV protease rabbit polyclonal antibody (Novus Biologicals, Cat. No. NBP1–97669) was used as the primary antibody at 1:500 dilution. An HRP-conjugated goat anti-rabbit antibody (Bio-Rad, Cat. No. 170–6515) was applied as the secondary antibody at 1:2000 dilution in all cases. Detection was performed with Western Lightning Plus ECL reagent (PerkinElmer, Cat. No. NEL104001EA) and imaged in a ChemiDoc MP imaging system (Bio-Rad).

100,000 cells were lysed directly in Laemmli buffer, lysates were vortexed and boiled for 5 min before loading into NuPage gels using the NuPage Gel Electrophoresis system at 200 V for 45 min. Gels were blotted onto Immobilon-FL PVDF membranes using the BioRad TransBlot® Turbo™ Semi-Dry transfer system. Primary antibodies (rat anti-α2 and rabbit anti-tubulin) were added at 4 °C overnight and the LI-COR secondary antibodies were added for 1 h. Membranes were visualised using a LI-COR Odyssey CLx Western Blot imaging system.