Rabbit anti ha

Confluent HFF monolayers grown on coverslips in 24-well plates were infected with freshly lysed parasites. Infected host cells were fixed with 4% paraformaldehyde in PBS, blocked for 30 min in 3% BSA+PBS, and permeabilized with 0.2% Triton X-100 (PBS-T) for 10 min. Primary antibodies (see below) diluted in 3% BSA-PBS were applied overnight at 4°C. Cultures were washed three times for 10 min each with PBS. Secondary antibodies diluted in 3% BSA-PBS were applied for 1hr at room temperature. Coverslips were washed three times for 10 min and 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, Life Technologies; #D1306) diluted 1:1,000 in PBS was applied for 10 mins at room temperature. Following three 10 min washes, coverslips were mounted using Vectashield antifade mounting medium (Vector Labs; #H-1000).
The following primary antibodies were used at the dilutions indicated: rat anti-HA (1:2,000; Roche #11867423001), rabbit anti-HA (1:2,000; Cell Signaling Technology #3724), mouse anti-TgF₁B-ATPase (1:4,000; ^{53 (link),72 (link)}), rat anti-TgSORTLR (1:2,000; ^{52 (link)}) and mouse anti-TgSERCA (1:2,000; ^{51 (link)}). Secondary antibodies conjugated to a fluorophore (Alexa Fluor; Thermo Fisher #A11005, #A11006, #A11007, #A11034) were used for IFAs at a 1:5,000 dilution.

Antibodies used in this work include Guinea pig anti Oc (1:750, gift from Tiffany Cook) (Xie et al., 2007 (link)); Rabbit anti Sox102F and Rabbit anti Slp1 (1:500 for both, Gifts from Claude Desplan); Rabbit anti-D (1:200) (from John R. Nambu), mouse anti-Ey (1:10, DSHB), sheep anti-GFP (1:500, AbD Serotec), Goat anti anti-beta-gal (Abcam 1:1000), rabbit anti-RFP (Abcam 1:1000), Rabbit anti HA (Cell Signaling Technology, 1:1000). Secondary antibodies are from Jackson or Invitrogen. Immunostaining was done as described (Li et al., 2013 (link)) with a few modifications: 3^rd instar Larval brains or adult brains were dissected in 1XPBS, and fixed in 4% Formaldehyde for 30 minutes on ice (larval) or 45min at RT (adult). Brains were incubated in primary antibody solution overnight at 4°C, washed three times and incubated in secondary antibody solution overnight at 4°C, washed three times and mounted in Slowfade. Images are acquired using a Zeiss Confocal Microscope. Figures are assembled using Photoshop and Illustrator.

Retinal tissues were prepared as described above. Antibodies used were as follows: chick and rabbit anti-GFP (1:500, Abcam; 1:5000, Millipore; 1:1000, AVES); goat anti-choline acetyltransferase (1:500, Millipore); goat anti-VAChT (1:1000, Millipore); goat anti-Sox2 (1:100, Santa Cruz); mouse anti-Isl1 (1:10, DSHB), rabbit anti-HA (1:500, Cell Signaling), and rabbit anti-DsRed (1:1000, Living Colors), and mouse anti-Ki67 (1:200, BD Biosciences). Nuclei were labeled with DAPI (1:1000, Invitrogen). Secondary antibodies were conjugated to Alexa Fluor 488, 568, and 647 (Invitrogen) and used at 1:1000. Fluoromount-G (SouthernBiotech) was used for mounting wholemounts. ProLong Gold Antifade was used for mounting retina section slides.

The following chemicals were used: HA-tagged ubiquitin-vinyl methyl ester, HA-UbVME (Enzo Life Science, Farmingdale, NY), forskolin (FSK) (Sigma-Aldrich), USP7 inhibitor HBX41108 (Tocris, Minneapolis, MN), USP10 inhibitor spautin-1 (Cayman, Ann Arbor, MI), cycloheximide (Sigma-Aldrich). All other chemicals were purchased from Sigma-Aldrich (St Louis, MO) or EMD Millipore (Billerica, MA), unless otherwise specified. Rabbit polyclonal anti-NHE3 antibody EM450 and mouse monoclonal anti-VSVG antibody P5D4 were previously described.^{20 (link),21 (link)} The following commercial antibodies were used: rabbit anti-HA (Cell Signaling), mouse anti-Ub (Santa Cruz), rabbit anti-USP7 (Cell Signaling), rabbit anti-USP10 (Cell Signaling), rabbit anti-Nedd4–2 (Abcam, Cambridge, MA), mouse anti-Rab5a (Cell signaling), mouse anti-Rab7 (Santa Cruz), rabbit anti-VSVG (Sigma), and mouse anti-β-actin (Sigma).