Anti p62 610832

ZM-FLAG and ZL-FLAG were cloned into a pHCMV vector, allowing the expression of MOPV or LASV Z matrix protein with a FLAG tag in the C-terminal position (inserted between the XmaL and BamHI restriction sites). The same constructions were made for ZM and ZL-mCherry plasmids. TAX1BP1 plasmids were engineered into pEGFP-C1, allowing the expression of GFP-tagged proteins for immunofluorescence experiments. eGFP-NDP52 was a kind gift from Dr Mathias Faure (CIRI, Lyon, France). The primary antibodies used were: anti-actin (A3854), anti-FLAG (A8592), anti-ATG5 (A0856), and anti-TAX1BP1 (HPA024432), all from Sigma-Aldrich (Saint Quentin Fallavier, France); anti-NDP52 (ab68588) from Abcam (Cambridge, MA, USA); anti-GAPDH (sc-25778, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p62 (610832, BD Biosciences, San Jose, CA, USA), and anti-Z (Agro-Bio, La Ferté Saint-Aubin, France). The secondary antibodies used for Western blotting were anti-mouse conjugated to peroxidase (111-035-174) and anti-rabbit conjugated to peroxidase (111-035-144), all from Jackson ImmunoResearch (Cambridge House, UK). The secondary antibody used for confocal microscopy was an anti-mouse conjugated to Alexa 555 (A21424) from Invitrogen (Carlsbad, CA, USA). The pharmacological agent used was chloroquine (C6628), purchased from Sigma-Aldrich.

Total proteins were extracted in Ripa buffer, and 30 ug of proteins was loaded for Western blot analyses, performed on SDS-PAGE gels under denaturing conditions. For the analysis of Beclin1, LC3 and p62 proteins, 12% bis-acrylamide gels were employed, whereas EGFR proteins were separated on Nu-PAGE 4–12% Bis-Tris Gel (Invitrogen™, Thermo Fisher Scientific, Waltham, MA, USA). Proteins were then electrotransferred onto a nitrocellulose membrane, and nonspecific protein binding was blocked using 10% Blotto-0.1% Tween PBS or 5% BSA-0.1% Tween PBS, respectively. Membranes were incubated with primary antibodies (anti-LC3, L8918, Sigma Aldrich; anti-p62, 610832, BD Biosciences; anti-Beclin1, ab207612, Abcam; anti-EGFR, #2232, Cell Signaling; anti-β-actin A2066 Sigma-Aldrich) diluted 1:1000 in the blocking solution, overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies (antimouse LNA931V, GE Healthcare, antirabbit LNA934V, GE Healthcare) were used to detect the primary antibodies, and blot density was quantified with ImageJ™ software (NIH, Bethesda, MD, USA). Protein band quantifications were normalized on the total protein band at 48 KDa (Ponceau Red band) or on β-actin, according to the most appropriate technique for the experiment.