Clone 3f10

Protein extraction was carried out as described (Ma et al., 2015). Twenty microliter samples were run on 13% SDS–PAGE gels and blotted on PVDF membranes using semi‐dry blotting. The membranes were subjected to immunoblotting using either anti‐HA peroxidase at a dilution of 1 : 3000 (clone 3F10; Roche) or anti‐Avr2 antibody (1 : 5000 diluted) (Ma et al., 2015). The secondary antibody goat‐anti‐rat (P31470, Pierce) was used at a 1 : 5000 dilution. Luminescence was visualized by ECL using BioMax MR film.

Western blot analysis was performed as previously described [27 (link)]. Polyacrylamide gel (12.5%) (e-PAGEL, ATTO Corporation, Tokyo, Japan) was used for the observation of light- induced phosphorylation of ROC15 (Figs 5B, S9B and S9C) and 7.5% polyacrylamide gel (e-PAGEL, ATTO Corporation) was used for the detection of HA-CETL (Figs 3B and S7E). The transfer step was performed using a Qblot kit, EZ blot kit and EZFastBlot HMW kit (ATTO Corporation)). BLOCK ACE Powder (KAC, Kyoto, Japan) was used to block the membranes. Rat monoclonal anti-HA antibody was used as the primary antibody (1:10000, clone 3F10, Roche, Basel, Switzerland). Horseradish peroxidase-conjugated goat anti-rat IgG was used as a secondary antibody (1:25000, Merck KGaA, Darmstadt, Germany).
Immunocytochemistry was performed as described previously [27 (link)]. Rat monoclonal anti-HA (1:1000) and mouse monoclonal anti-NPC (1:1000, clone MAb414, Labcorp, NC, USA) were used as primary antibodies. Alexa Fluor 488-conjugated goat anti-rat IgG (Thermo Fisher Scientific) and Alexa Fluor 647-conjugated goat anti-mouse IgG (Thermo Fisher Scientific) were used as secondary antibodies. Fluorescence was observed using a laser scanning confocal fluorescence microscope (FV10i-DOC; Olympus, Tokyo, Japan).

Western blot analysis was performed as described previously [21 (link)]. We used rat monoclonal anti-HA antibody (1:10,000–1:50,000; clone 3F10, Roche), mouse monoclonal anti-HA antibody (1:1,000; COVANCE, clone 16B12), and mouse monoclonal anti-FLAG antibody (1:1,000; Sigma, clone M2) as the primary antibody. Horseradish peroxidase (HRP) conjugated antibodies were used as the secondary antibody.

Transiently transfected HEK293T cells were seeded in a poly-L-lysine coated 96-wells plate for 48 h at 37 °C and 5% CO₂. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.5% NP-40 as described above. In the case of determination of surface receptor expression levels, cells were only fixed with 4% paraformaldehyde. Receptor expression was detected using a Rat anti-HA antibody (1:1000 in 1% (v/v) FBS/PBS, Clone 3F10, Roche) and HRP-conjugated Goat-anti-Rat antibody (1:1000 in 1% (v/v) FBS/PBS, Pierce). Detection of intrabody-FLAG expression was done using a Mouse-anti-FLAG antibody (1:1000 in 1% (v/v) FBS/PBS, Clone M2, Sigma-Aldrich) and HRP-conjugated Goat-anti-Mouse antibody (1:2000 in 1% (v/v) FBS/PBS, Pierce). In the case of U251-iUS28 cells, receptor expression was detected using an Rabbit-anti-US28 antibody (1:1000 in 1% (v/v) FBS/PBS, Covance^{24 (link)}) and HRP-conjugated Goat-anti-Rabbit antibody (1:1000 in 1% (v/v) FBS/PBS, Bio-Rad). Incubation with antibodies was done for 1 h at RT. Wells were washed three times with 1× PBS in between all incubation steps. Antibody binding was detected using 1-Step ultra TMB-ELISA substrate (Thermo Fisher Scientific) and the reaction was stopped with 1 M H₂SO₄. Optical density was measured at 450 nm with a PowerWave plate reader (BioTek). Data were analyzed using GraphPad Prism version 8.0.

The in vivo Tip1 ubiquitination assay was performed as previously described^{61 (link)}, except using the vector pREP1-6His-myc-ubiquitin instead of pREP1-6His-ubiquitin for expressing ubiquitin-fusion proteins. Briefly, Tip1-6HA strains transformed with pREP1-6His-myc-ubiquitin were grown in the absence of thiamine for 20–24 h to induce 6His-myc-ubiquitin expression. Samples were collected and extracts were prepared in lysate buffer (8 M urea, 300 mM NaCl, 50 mM Na₃PO4, 0.5% NP-40, 4 mM imidazole). Ubiquitin conjugates were purified on Ni-NTA beads and washed by washing buffer (8 M urea, 300 mM NaCl, 50 mM Na₃PO4, 0.5% NP-40, 20 mM imidazole) for four times, separated on SDS-PAGE, and immunoblotted with rabbit polyclonal anti-Myc (GeneScript, A00172-40) (1:1000) to detect ubiquitin and rat monoclonal anti-HA (clone 3F10; Roche, Cat. No. 11867423001) (1:1000) to detect ubiquitinated Tip1-6HA.

The CHIP assay was manipulated as previous method using 100 mg of tobacco root with or without HT stress [31 (link)]. Immunoprecipitation was performed with rabbit polyclonal antibody against HA (1:1000, clone 3F10, Roche). NtPMT1 region were detected using the PCR primers listed in Table S1. Chip products were analyzed by quantitative PCR. Data from the Chip experiments are expressed as the mean ± SD of three biological replicates.