Mouse anti myc 9e10

Cell extracts for expression analysis were prepared as described (14 (link)). Briefly, yeast were grown in selective SD media to mid-exponential phase, whereafter 2.5 A₆₀₀ units were harvested and lysed by an alkaline lysis method (45 (link)). Lysates were resuspended in SDS sample buffer and incubated at 37 °C for 30 min, centrifuged to remove cell debris, and analyzed by SDS-PAGE and immunoblotting.
Immunoblotting was performed as described (46 (link)) with the following antibodies: rabbit anti-Doa10 at 1:2000 (27 (link)); mouse anti-MYC (9E10, Covance) at 1:10,000; rabbit anti-G6PDH (A9521, Sigma) at 1:10,000; mouse anti-FLAG (F3165; Sigma) at 1:2000 or 1:10,000; peroxidase anti-peroxidase (Sigma) at 1:1000; mouse anti-PGK (459250, Thermo Fisher Scientific) at 1:20,000; rabbit anti-Dfm1 at 1:2000 (47 (link)); rabbit anti-Ubc6 at 1:2000 (31 (link)). Primary antibody incubations were followed by peroxidase-coupled sheep anti-mouse or peroxidase-coupled goat anti-rabbit secondary antibodies (GE Healthcare) at 1:10,000 and visualized by enhanced chemiluminescence (48 (link)) on a G:Box system (Syngene).

PLA detection was carried out using the Duolink In Situ Red Fluorescence kit (Sigma-Aldrich) according to the manufacturer's instructions. Briefly, PFA-fixated HEK293T cells expressing Flag-EBNA-LP and myc-tagged-RBM4 were blocked for 1 h at room temperature. Primary antibodies were added at a dilution of 1:200 (mouse anti-myc 9E10, Covance, Inc.) and 1:500 (rabbit anti-Flag, Sigma-Aldrich) in 40 μl Duolink antibody diluent and incubated at 4°C overnight. Samples were washed twice with Wash Buffer A for 5 min each, then secondary antibodies (Duolink anti-mouse PLA-minus probe and Duolink anti-rabbit PLA-plus probe) were added for 1 h at 37°C. After two washes with Wash Buffer A, ligation mix was added, for 30 min at 37°C and the samples washed again twice with Wash Buffer A. The amplification reaction was carried out at 37°C for 100 min. Slides were subsequently washed twice with 1 x Wash Buffer B, once with 0.01× Wash Buffer B and mounted with Duolink In Situ Mounting Medium with DAPI. Microscopic examination was performed using an AxioImager Z1 epifluorescence microscope (Zeiss). Images were analysed using FIJI/ImageJ software.

The following primary antibodies are used: Mouse anti-Actin (Millipore, MAB 1510), Mouse anti-Myc 9E10 (Covance, MMs-150R), Mouse anti-Ubiquitin (Santa Cruz, sc-8017), Mouse anti-GFP (Roche, 11814460001), Rabbit anti-ZDHHC6 (Sigma, SAB1304457), Mouse anti-Transferrin Receptor (Thermo Scientific, 136800), Mouse anti-Flag M2 (Sigma, F3165), Rabbit anti-ANTXR1 (Sigma, SAB2501028), Rabbit anti-TRAPα (Abcam, ab133238), Rabbit anti-Flotillin1 were produced in our laboratory, Mouse anti-CLIMP63 (Enzo, ALX-804–604), Mouse anti-Calnexin (MAB3126), Rabbit anti-GP78 AMFR (Abnova, PAB1684), Rabbit anti-IP3R (Cell signaling, 85685). The following beads were used for immunoprecipitation: Protein G Sepharose 4 Fast flow (GE Healthcare, 17-0618-01), anti-Myc affinity gel (Thermo Scientific, 20169), anti-Flag affinity gel EZview M2 (Sigma, F2426). Drugs were used as follows: Bafilomycin A1 at 100 nM (Sigma, B1793), MG132 at 10 µM (Sigma, C2211), Hydroxylamine at 0.5 M (Sigma, 55459), mPEG-5k at 20 mM (Sigma, 63187), N-ethylmaleimide NEM at 20 mM (Thermo Scientific, 23030), Tris-2- carboxyethyl-phosphine hydrochloride TCEP at 10 mM (Thermo Scientific, 23225), Methyl methanethiosulfonate MMTS at 1.5% (Sigma, 208795), Puromycin at 3 µg/ml (Sigma, P9620).