Ab24586

Unconditioned medium or sickle or control MCCM-treated mBECs cultured in eight well chamber slides (ibidi USA, Madison, WI, USA) were fixed with 4% paraformaldehyde, permeabilized with 0.05% Triton X-100, for 10 min, washed with PBS containing 0.01% Tween-20 (PBS-Tw) and blocked by incubation in 3% normal donkey serum/PBS-Tw. The mBECs were incubated for 1 h at room temperature with primary antibodies diluted in 3% normal donkey serum/PBS-Tw. The following antibodies were used: ER-specific rabbit anti-galactose-1-phosphate uridylyltransferase antibody (GalT, 1:100; ab178406, Abcam, Cambridge, MA, USA), Golgi-specific rabbit anti-Giantin antibody (1:100; ab24586; RRID:AB_448163, Abcam), goat anti-P-selectin (1:100; AF737; RRID:AB_2285644, R&D, Minneapolis, MN, USA). Slides were washed and then incubated for 1 h at room temperature with species-specific donkey secondary antibodies conjugated with Cy2 and Cy3 (1:200; Jackson ImmunoResearch Laboratories, West Grove, PA, USA) diluted in 3% normal donkey serum/PBS-Tw to visualize the immunoreactive proteins. Samples were mounted with Vectashield (H-1000, Vector Labs, Burlingame, CA, USA) and fluorescence images were captured using Olympus IX 70 inverted microscope (Olympus Corporation, Center Valley, PA, USA).

All of the individually sourced antibodies for western blotting (WB), immunofluorescence and the LPAAT assay were obtained and used as detailed in ref. 15 (link), unless otherwise stated. The rabbit polyclonal anti-LPAATδ (ab188002, 1:1,000) and anti-giantin (ab24586, 1:5,000) antibodies (for WB use) were from Abcam; the mouse polyclonal anti-LPAATδ antibody (for microinjection and LPAAT assay use) was from Abnova (H00056895-B01P); the rabbit polyclonal anti-LPAATδ antibody (1:50 for IF use) was from Sigma-Aldrich (SAB4502436), the anti-GM130 monoclonal antibody (1:200 for IF use) was from BD Transduction Laboratories (610823) and the rabbit polyclonal anti-Calnexin antibody (1:500 for WB use) was from Santa Cruz (AF18 sc23954). The anti-LPAATδ polyclonal antibodies from Abcam and Abnova recognize specifically LPAATδ and not other LPAATs by WB analysis (our unpublished data). Commercially available antibodies used: anti-TGN46 1:400 for IF use (Biorad, AHP500GT); anti-Giantin 1:500 for IF use (Alexis Corporation, 804-600-C100); anti-Flag 1:5,000 for WB and 1:500 for IF use (Sigma, F1804); anti-GAPDH 1:50,000 for WB use (ABD Serotec, MCA4740); anti-penta-His 1:1,000 for WB use (Life Technologies, P21315); anti-LPAATγ 1:1,000 for WB use (Abcam, ab93181). Source images from relevant WB are available in the Supplementary Figs 11–13.