Pm036

Primary antibodies for western blot were against LC3 (Novus Biologicals, NB600), SQSTM1 (Abcam, ab56416), ATG13 (Sigma Aldrich, SAB4200100) and ACTIN (EMD Millipore, MAB1501). Primary antibodies used for immunofluorescence and immunocytochemistry were against LC3 (MBL, PM036), SQSTM1 (Progen, GP62-C), ATG16L1 (Abgent, AP1817b), AQP5 (Alomone labs, AQP5-005) and BrdU (Bio-Rad, MCA2483GA). Fluorescently-labeled antibodies for FACS sorting were against PECAM1/CD31 (PECAM1/CD31-PE; eBioscience, 12–0311-82), PTPRC/CD45 (PTPRC/CD45-PE, Biolegend, 103106), LY76/TER119 (LY76/TER119-PE/Cy7; Biolegend, 116222), CD24 (CD24-Pacific Blue, Biolegend, 101820) and ITGB1/CD29 (ITGB1/CD29-FITC, BD Biosciences, 555005). The following secondary antibodies from ThermoFisher Scientific were used for the visualization of the primary antibodies: Alexa Fluor 488-conjugated goat anti-mouse (A-11001), Alexa Fluor 568-conjugated goat anti-mouse (A-11031) or goat anti-rabbit (A-11011), Alexa Fluor 647-conjugated goat anti-mouse (A-21235), and Alexa Fluor 568 conjugated goat anti-Guinea pig (A-11075).
Hoechst 33342 was from Sigma Aldrich (B2261), Chloroquine (C6628) was from Sigma Aldrich and bafilomycin A₁ was from BioAustralis (BIA-B1012). Tat-beclin 1 was from Selleck Chemicals (S8595), while propidium iodide was from Sigma Aldrich (P4170).

40 x 10⁴ SH-Sy5y cells, were seeded in 24-well culture plates on coverslips. Cells were treated as specified for the specific experiments. Cells were fixed with 4% formaldehyde (VWR 9713.1000) for 15–20 min at RT, and permeabilized with methanol for 10 min at −20°C and blocked in PBS 3% donkey serum for 1 h at RT. The fixed cells were stained with rabbit anti-LC3 (Nordic Biosite, PM036, dilution 1:2000), guinea pig anti-p62 (Progen, GP62-C, dilution 1:200), rabbit anti-β3-tubulin (Cell Signaling, 2146, 1:100) for 1 h at RT. The cells were washed three times with PBS and incubated for 1 h with a secondary antibody of donkey anti-rabbit Alexa 488 antibody (Thermo Fischer, A-21206, dilution 1:1000) for LC3, a secondary goat anti-guinea pig Alexa A568 (Thermo Fischer, A-11075) for P62 and DAPI. Antibodies were diluted in PBS 1% donkey serum. After three washes with PBS, coverslips were mounted using Prolong Gold mounting medium (Thermo Fischer P36934). Images were acquired on a Zeiss LSM 800 confocal microscope and processed with the Fiji software package. LC3 foci were counted manually in 25 randomly selected cells per condition. HSV1 capsids associated with the nuclear rim were quantified over 100 nuclei.