Anti flag m2 antibody f3165

The following antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): anti-OGT (sc-74546), anti-E6AP (sc-166689), anti-hemagglutinin (HA) (sc-7392), anti-actin (sc-8432), anti-UB (sc-8017), and mouse anti-rabbit IgG-HRP (sc-2357). Goat anti-mouse IgG secondary antibody (31438) was from Thermo Fisher Scientific (Waltham, MA, USA). Anti-FLAG (M2) antibody (F3165) was from Sigma-Aldrich (Burlington, MA, USA). Anti-Nup62 antibody (NBP1-31381) was from Novus Biologicals (Centennial, CO, USA). Anti-Sp1 antibody (07-645) was from EMD Millipore (Burlington, MA, USA). Anti-O-GlcNAc RL2 antibody (2739) was from Abcam (Cambridge, MA, USA). E6 protein of human papillomavirus type 16 was from Boston Biochem (Cambridge, MA, USA).

The following antibodies were used throughout this study: From EMD Millipore, Anti-Influenza A HA (AB1074) (1:200), FITC Anti-Influenza A Nucleoprotein clone A1 (MAB8257F) (1:200). From Abcam, Anti-LAMP1 clone H4A3 (ab25630) (1:100), Anti-Rab7 Alexa-Fluor647 clone EPR7589 (ab198337) (1:100), Anti-ATP6V0D1 (ab56441) (1:2000), β-actin antibody (ab6276) (1:10000). From BD bioscience, FITC mouse anti-human CD71 (555536) (1:100). From Thermofisher, Alexa-Fluor488 Goat anti-mouse IgG (1:500), Alexa-Fluor488 Donkey anti-goat IgG (1:500). From Sigma Aldrich, Anti-Flag M2 antibody (F3165) (1:2000). From Cell Signaling Technology, TFEB antibody (#4240S) (1:2000), Phospho-TFEB antibody (Ser211) (#37681S) (1:2000), Cox-IV antibody (4850s) (1:2000), HSP90 antibody (#4874) (1:2000) and TBP antibody (#8515) (1:2000). From Abnova, Anti-ATP6V1A (H00000523-A01) (1:2000).

Affinity purification–mass spectrometry (AP–MS) identification of binding partners was performed as described previously [69 (link)]. Notably, 293T cells transfected with Flag-NUSAP1 were lysed in 0.2% NP-40 buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM CaCl₂, 1 mM EDTA, 0.2% NP-40, Halt protease, and phosphatase inhibitor). Lysates were homogenized 20 times using a Dounce homogenizer (pestle B) (Kimble Chase) and incubated with micrococcal nuclease (Thermo Scientific, Waltham, MA, USA). Cell extracts were collected by centrifugation at 14,000 rpm at 4 °C for 20 min. Cell extracts were incubated with antibodies (anti-Flag M2 antibody, F3165, Sigma Aldrich, Saint Louis, MO, USA) overnight at 4 °C with rotation. The next day, Protein-G Dynabeads (Invitrogen) were added to the mixture and incubated for 3 h at 4 °C with rotation. After incubation, the beads were washed three times in ice-cold wash buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40). Proteins were eluted in acid using IgG elution buffer (Thermo Scientific) at room temperature (RT) for 10 min with gentle vortexing. The final elution was collected and neutralized with 1/10 volume of 1 M Tris-HCl, pH 9.0.