Nb600 363

293T cells in 6-wells were transfected with 1 ug of expression constructs or empty pcDNA3 vector for a total of 2 ug per well, using BioT transfection reagent (Bioland) according to manufacturer protocol. At 24 hours post-transfection, cells were lysed in cold lysis buffer (1% NP-40, 100 mM Tris-HCl pH 7.5, 150 mM NaCl, 5% glycerol, 1 mM EDTA, 1X protease inhibitor cocktail (Roche)), clarified at 14k rpm for 5 minutes at 4°C, and incubated with 3 ug of rabbit anti-NiV-M [50 (link)] or rabbit anti-HA (Novus, NB600-363) at 4°C overnight. Following incubation with protein G agarose (Pierce) for 2 hours at 4°C, the bound protein was washed 4 times with cold wash buffer (same composition as lysis buffer but with 0.2% NP-40), then eluted in reducing Laemmli SDS sample buffer at 95°C.

An antibody against PDGFRα (AF 1602) was purchased from R&D Systems (Minneapolis, MN, USA). The antibodies recognizing phospho-PDGFRα (pY849, #3170), phospho-PLCγ1 (pTyr783, #2821), PLCγ1 (#2822), phospho-STAT3 (pTyr705, D3A7, #9145), STAT3 (79D7, #4904), phospho-Akt (pSer473, D9E, #4060), Akt (#9272S), phospho-ERK1/2 (pThr202/pThr204, #9101) and ERK1/2 (137F5, #4695) were from Cell Signaling Technology (Danvers, MA, USA). Antibodies against SUMO1 (sc-5308) and ubiquitin (sc-8017) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The α-tubulin antibody (T6074) was from Sigma-Aldrich (St Louis, MO, USA). Primary antibodies against HA (NB600-363) and the transferrin receptor (TfR) (NB100-92243) were from Novus Biologicals (Centennial, CO, USA). Antisera recognizing Alix (HP95) [42 ] and PDGFRβ (CTβ) [43 ] were prepared in house. The secondary antibodies used for immunoblotting included goat anti-mouse IgG (62-6520), goat anti-rabbit IgG (65-6120) and rabbit anti-goat IgG (81-1620), were from Invitrogen (Waltham, MA, USA). The inhibitors MG132 (C2211) and chloroquine (C6628) were from Sigma-Aldrich (St Louis, MO, USA). The inhibitor ginkgolic acid (345,887) was purchased from Merck KGaA and bortezomib (#2244) was from Cell Signaling Technology (Danvers, MA, USA).

The following commercial antibodies were used: SFPQ-specific mouse monoclonal antibody [sc-374502, Santa Cruz Biotechnologies, Dallas, TX, USA; western blotting (WB) 1:500, immunocytochemistry (ICC) 1:100], anti-HA tag rabbit polyclonal antibody (NB600-363, Novus Biologicals, Centennial, CO, USA; WB 1:1000, ICC 1:500), pGA repeat rabbit polyclonal antibody (24492-1-AP, Proteintech, Rosemont, IL, USA), pGR repeat rabbit polyclonal antibody (23978-1-AP, Proteintech), pGP repeat rabbit polyclonal antibody (24494-1-AP, Proteintech), pAP repeat rabbit polyclonal antibody (24493-1-AP, Proteintech) and pPR repeat rabbit polyclonal antibody (23979-1-AP, Proteintech). All DPR antibodies were used at a 1:1000 dilution for dot blots, including GAPDH rabbit polyclonal antibody (10494-1-AP, Proteintech), GAPDH mouse monoclonal antibody (60004-1-Ig, Proteintech), anti-mouse IgG (H+L), F(ab′)2 Fragment (Alexa Fluor 488 Conjugate) (4408, Cell Signaling Technology, Danvers, MA, USA; dilution 1:1000), StarBright Blue 520 Fluorescent Secondary Antibodies (nos. 12005866 and 12005869, Bio-Rad, Hercules, CA, USA; dilution 1:5000), StarBright Blue 700 Fluorescent Secondary Antibodies (nos. 12004158 and 12004161, Bio-Rad; dilution 1:5000) and Peroxidase AffiniPure Goat Anti-Rabbit IgG (H+L) (111-035-045, Jackson ImmunoResearch, West Grove, PA, USA; dilution 1:5000).