Anti c myc

α-sarcin was purchased from Santa Cruz. ³⁵S methionine and ³⁵S cysteine were purchased from Perkin Elmer. Alexa FluorA647 succinimidyl ester was purchased from ThermoFisher Scientific. Inhibitors were purchased from commercial vendors as follows: Aim-100 (Apexbio), Wiskostatin (Sigma), Dynasore (Sigma), Pitstop-2 (Sigma), CK-869 (Sigma), Pirl1 (Hit2leads). Anti-EMCV mouse polyclonal antibodies were provided by Michael Diamond. Anti-poliovirus antibodies were provided by Nihal Altan-Bonnet. Other antibodies were obtained from commercial vendors as follows: Anti-coxsackie virus B3 antibodies (ThermoFisher), Anti-adenovirus A5 antibodies (ThermoFisher), Anti-influenza A virus NP antibodies (Millipore), Anti-TNK2 (A11) (Santa Cruz), Anti-WASL (Abcam and Sigma), Anti-actin, clone C4 (Sigma), Anti-NCK1 (Millipore), enterovirus D68 Ab (GeneTex), Anti-HA (ThermoFisher), Anti-Flag (GenScript), Anti-c-Myc (Invitrogen), Anti-double stranded J2 antibodies (Scicons).

Immunoblots were performed as previously described [50 (link)]. The following antibodies were used: anti-AKT1 (2H10), anti-AKT2 (L79B2), anti-AKT3 (L47B1), anti-mTOR (7C10) and anti-AMPK-alpha (2B7), purchased from Cell Signaling (Danvers, MA, USA); anti-HADHA, anti-VDAC1, anti-HARS2 and anti-SSBP1 obtained from Abcam (Cambridge, UK); anti-cMYC purchased from Invitrogen; anti-β-actin and anti-LC3B obtained from Sigma; and HRP anti-mouse and anti-rabbit antibodies purchased from Dako (Jena, Germany). Densitometry readings were performed with ImageJ (SYBYL Project, Newcastle, UK) by normalizing with background.

PC12 (rat adrenal gland phaeochromocytoma) cells were grown at 37°C and 5% CO₂ in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Carlsbad, CA) with 20% fetal bovine serum (FBS; Atlanta Biologicals, Flowery Branch, GA), 100 units/ml penicillin and 100 units/ml streptomycin (Life Technologies, Carlsbad, CA). pcDNA3.1mycC expressing human huntingtin fragment (1–90) containing 73 polyglutamine repeats (Coriell Institute; CHDI-90000034) was used for stable transfection. Cells were seeded to 70% confluency and grown overnight. 15 µl of Attractene Transfection Reagent (Qiagen, Gaithersburg, MD) was added to 4 µg plasmid DNA diluted in 300 µl Opti-MEM (Life Technologies, Carlsbad, CA). Cells were grown in complete media and selected for four weeks using 500 mg/ml G418 (Life Technologies, Carlsbad, CA). To create monoclonal cultures, single colonies were isolated using dilution cloning, picked with filter paper, grown in a 6-well plate and screened for transgenic expression by Western blot analysis using mouse Anti- c-Myc (Novex, R950-25, Life Technologies, Carlsbad, CA).

To further confirm whether the ARV p17 protein interacts with PQBP1, IGF2BP1 or FGF1, co-IP assays were carried out. After cotransfection of p17-Flag with recombinant vectors expressing PQBP1-Myc, IGF2BP1-Myc or FGF1-Myc for 48 h in DF-1 cells, immunoprecipitation was performed using an anti-c-Myc or anti-Flag agarose affinity gel (Thermo Fisher Scientific, 23620, Waltham, MA, USA) according to the manufacturer’s protocol. Briefly, cells were scraped from the culture plate and lysed with a mixture of RIPA buffer (Beyotime, Beijing, China) containing PMSF. After centrifugation for 20 min at 4 °C, the supernatant was collected, mixed with 10 μL of agarose slurry and incubated overnight at 4 °C. The protein agarose mixture was washed 3 times with TBST and resuspended in 2× nonreducing sample buffer. Finally, the liquid was heated for 5 min, with 2 μL of 2-ME added to the samples during denaturation, followed by analysis by Western blotting with specific antibodies.