Ma5 15256

Seedlings were homogenized in extraction buffer (150 mM Tris–HCl, pH 7.5, 6 M urea, 2% SDS and 5% μ-mercaptoethanol), boiled for 5 min and cell debris removed by centrifugation at 18 000 × g at 4°C for 10 min. The supernatants were resolved on 12% SDS-PAGE, transferred to Hybond PVDF membranes (GE Healthcare) and subjected to western blot analysis. Antibodies used for detection: for 3xHA-tagged HsfA1a, HRP-conjugated antibody (3F10, Roche); for GSy tag (52 (link)), anti-GFP antibody (MA5-15256, Thermo Fisher); for sHSP-CI, anti-sHSP17.6 antibody (AS07 254, Agrisera), anti-HSP70 antibody (AS08 371, Agrisera), anti-HSP90-1 antibody (AS08 346, Agrisera), anti-HSP101 (AS07 253, Agrisera), anti-Ub antibody (U5379, Sigma-Aldrich) and anti-SUMO1 antibody (AS08 308, Agrisera); as secondary antibody, we used monoclonal HRP-conjugated anti-mouse (A4416, Sigma-Aldrich) or anti-rabbit (A6154, Sigma-Aldrich). The proteins were visualized by chemiluminescence (ECL kit; GE Healthcare) and quantified by Image Lab 5.1 (Bio-Rad); protein signals have been normalized to Rubisco large subunit (RbcL), having a slow turnover rate (67 (link)).

To measure the expression level of Doa10, 2.0 OD₆₀₀ units of cells were collected, resuspended in 230 μl of 0.26 M NaOH and 0.13 M β-mercaptoethanol. Cell suspensions were incubated at room temperature for 5 min, then spun down by centrifugation at 6000 g for 3 min. Pellets were resuspended with 50 μl of reduced SDS sample buffer. Cell lysates were incubated at 37°C for 30 min and clarified by centrifugation at 21,000 g for 10 min before analysis by SDS-PAGE. SDS-PAGE was performed using Tris-glycine gels, except for Fig.5b,c where 4-12% Bis-Tris SDS-PAGE gels (Thermo Fisher) were used.
Immunoblotting experiments were performed with anti-Doa10 antiserum (a gift from M. Hochstrasser; 1:1,000 dilution), anti-GFP antibody (Thermo Fisher #MA5-15256 ; 1:3,000 dilution), anti-ALFA-tag nanobodies fused with a rabbit Fc domain (home-made), anti-Strep-tag antibody (Genscript #A01732, ; 1:2,000 dilution), anti-FLAG-tag antibody (Sigma #F1804; 1,1000 dilution), anti-Pgk1 antiserum (a gift from J. Thorner; 1:1,000 dilution). Secondary antibodies used in this study were goat anti-rabbit ( Thermo Fisher #31460; 1:10,000 dilution), goat anti-mouse (Thermo Fisher #31430; 1:10,000 dilution).

N. benthamiana leaves were grinded in liquid nitrogen, and 1 g of leaf powder was dissolved in a 2-mL extraction buffer GTEN (10% glycerol, 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA) freshly mixed with 2% (w/v), polyvinylpolypyrrolidone, 1X protease inhibitor, (Thermo #88666) and 10 mM dithiothreitol, 0.1% (v/v) Tween 20. The cell debris was pelleted by centrifugation and supernatant was collected in a fresh tube (first with 3,000 g for 10 min, and again twice with 21,000 g for 10 min in microcentrifuge at 4 °C). For western blot analysis, anti-FLAG (Thermo MA1-91878) and anti-GFP (Thermo MA5-15256) antibodies were used as the primary, and anti-mouse Alkaline Phosphatase conjugated antibody (Chemicon International #AP308A) was used as the secondary antibody.

The immunostaining was analyzed as previously described [30 (link)]. Briefly, cells were plated and grown on glass coverslips for 48 h, fixed for 10 min with 4% PFA at RT and then stored in PBS with 0.05% sodium azide at 4 °C until they were immunostained. Briefly, the cells were permeabilized for 3 min at 4 °C in PBS containing 0.5% Triton X‐100 and the coverslips were then probed overnight at 4 °C with the primary antibody against Ki67 (1 : 200, #PA1‐21520: Thermo Scientific), POTEE (1 : 500, #SAB1301635: Sigma‐Aldrich), Rac1 (1 : 200, #610650: BD Biosciences), Phalloidin (Phalloidin‐iFluor 594, #ab176757: Abcam), Cortactin (1 : 200, #ab33333: Abcam) or green fluorescent protein (GFP, 1 : 1000, #MA5‐15256: Thermo Scientific). After rinsing thoroughly, the coverslips were incubated with Alexa Fluor 488, 594, or 635 goat anti‐mouse‐IgG or anti‐rabbit‐IgG secondary antibodies (Invitrogen), counterstained with DAPI and then mounted on glass slides using the Gold antifade reagent (#P36935, Invitrogen). Immunofluorescence was analyzed under a Zeiss microscope (Zeiss Axioplan 2) and to calculate the proliferation ratio using the Ki67 marker, we counted the total number of positively stained tumor cells in each image/field and the total number of tumor cells in each image. The percentage of Ki67⁺ cells was calculated as: No. of positive tumor cells/total No. of tumor cells × 100.

To measure the expression level of Doa10, 2.0 OD₆₀₀ units of cells were collected, resuspended in 230 µl of 0.26 M NaOH and 0.13 M β-mercaptoethanol. Cell suspensions were incubated at room temperature for 5 min, then spun down by centrifugation at 6000 g for 3 min. Pellets were resuspended with 50 µl of reduced SDS sample buffer. Cell lysates were incubated at 37 °C for 30 min and clarified by centrifugation at 21,000 g for 10 min before analysis by SDS-PAGE. SDS-PAGE was performed using Tris-glycine gels, except for Fig. 5b, c and Fig. 6e, f where Bis-Tris SDS-PAGE gels were used.
Immunoblotting experiments were performed with anti-Doa10 antiserum (a gift from M. Hochstrasser; 1:1,000 dilution), anti-GFP antibody (Thermo Fisher #MA5-15256; 1:3,000 dilution), anti-ALFA-tag (ref. ^{71 (link)}) and anti-SPOT-tag (BC2; ref. ^{72 (link)}) nanobodies fused with a rabbit Fc domain (home-made), anti-Strep-tag antibody (GenScript #A00626; 1:2,000 dilution), anti-FLAG-tag antibody (Sigma #F1804; 1,1000 dilution), anti-Pgk1 antiserum (a gift from J. Thorner; 1:1,000 dilution). Secondary antibodies used in this study were goat anti-rabbit (Thermo Fisher #31460; 1:10,000 dilution), goat anti-mouse (Thermo Fisher #31430; 1:10,000 dilution).