Mouse anti ubiquitin antibody

COS-7 cells and HEK293 cells were purchased from ATCC (Manassas, VA, USA). [³H]-labeled estrone sulfate (ES) and [³H]-labeled p-aminohippuric acid (PAH) were ordered from PerkinElmer (Waltham, MA, USA). Mouse anti-Myc antibody (9E10) was purchased from Roche (Indianapolis, IN, USA). Mouse anti-E-Cadherin antibody was from Abcam (Cambridge, MA, USA). Streptavidin agarose resin, protein G agarose, and Sulfo-NHS-SS-biotin were ordered from Thermo Scientific (Rockford, IL, USA). The 20S proteasome assay kit was ordered from Cayman Chemical Company (Ann Arbor, MI, USA). Mouse anti-β-actin antibody, normal mouse IgG, and mouse anti-ubiquitin antibody were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). Ixazomib, oprozomib, and delanzomib were purchased from Selleck Chemicals (Houston, TX, USA). Probenecid, lactacystin, epoxomicin and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Parental COS-7 cells and HEK293 cells were purchased from ATCC (Manassas, VA, USA). Hydroxychloroquine Sulfate and Chloroquine Disulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Membrane-impermeable biotinylation reagent sulfo-NHS-SS-biotin, streptavidin agarose, protein G-agarose beads, and horseradish peroxidase-conjugated anti-mouse antibodies were obtained from Pierce Biotechnology (Rockford, IL, USA). We purchased [³H]-labeled estrone sulfate from PerkinElmer (Waltham, MA, USA). Mouse anti-myc antibody (9E10) was purchased from Roche (Indianapolis, IN, USA). Mouse anti-E-cadherin antibody was purchased from Abcam (Cambridge, MA, USA). Mouse anti-ubiquitin antibody, mouse anti-β-actin antibody and normal mouse IgG were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). A 20S proteasome assay kit was purchased from Sigma-Aldrich (St. Louis, MO, USA). Normal mouse lgG, mouse monoclonal anti-ubiquitin and mouse monoclonal anti-β-actin antibodies were purchased from Santa Cruz (Santa Cruz, CA, USA).

A549 cells were infected with hPIV1 or hPIV3 at an MOI of 5 and cultured for 2 days. Washed cells were labeled with ³⁵S-Met/Cys for 30 min and chased for either 2 or 4 h with DMEM-8% FCS. HMGCR in the cell lysates was immunoprecipitated using anti-HMGCR Ab coupled to Protein G Dynabeads. To analyze HMGCR degradation post infection, cells were infected at MOI 5 for 8 h and treated with proteosomal degradation inhibitor MG132 (10 μM) or lysosomal degradation inhibitor NH₄Cl (20 mM) for an additional 8 h and HMGCR protein levels were analyzed by immunoblotting. For the analysis of HMGCR ubiquitination, cells infected with hPIV1 or hPIV3 at an MOI of 3 were cultured for 18 h at 34°C. MG132 (10 uM) or DMSO was added and cells were cultured for an additional 8 h. HMGCR in the cell lysates was immnoprecipitated as described above and analyzed for ubiquitination by Western blotting using mouse anti-ubiquitin antibody (Santa Cruz).