Pi4kiiiβ

Vero and QM5 cells were maintained at 37˚C and 5% CO₂ in medium 199 with Earle's salts supplemented with 5 or 10% fetal bovine serum (FBS), respectively_. The rabbit polyclonal anti-p14 and anti-14 ectodomain antisera were previously described (Corcoran and Duncan, 2004 (link); Top et al., 2005 (link)). Antibodies against actin (Sigma-Aldrich, St. Louis, MO), myc epitope tag (Sigma-Aldrich), PI4KIIIβ (BD Biosciences, Franklin Lakes, NJ), TGN46 (Serotec, Oxford, UK), protein disulfide isomerase (PDI; Enzo Life Sciences, Farmingdale, NY), horseradish peroxidase (HRP)–conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA), Alexa Fluor 488–conjugated goat anti-mouse or donkey anti-sheep, Alexa Fluor 555–conjugated donkey anti-rabbit, and Alexa Fluor 647–conjugated goat anti-rabbit secondary antibodies (Life Technologies, Carlsbad, CA) were obtained from the indicated suppliers.

HeLa cells were maintained in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Life Technologies). HEK cells were maintained in DMEM supplemented with 10% FBS and 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Life Technologies). Rabbit polyclonal anti-p14 and anti-p14 ectodomain (anti-p14ecto) antibodies were previously described (Corcoran and Duncan, 2004 (link); Top et al., 2005 (link)). Antibodies against actin (Sigma-Aldrich, St. Louis, MO), Rab11A (BD Biosciences, Franklin Lakes, NJ), AP-1γ (Sigma-Aldrich), AP-3δ (Developmental Studies Hybridoma Bank, Iowa City, IA), AP-4ε (Abcam, Cambridge, MA), PI4KIIIβ (BD Biosciences), TGN46 (AbD Serotech, Oxford, UK), horseradish peroxidase (HRP)–conjugated goat anti-rabbit (Jackson ImmunoResearch, West Grove, PA), HRP-conjugated goat anti-mouse (Santa Cruz Biotechnology, Dallas, TX), Alexa 488–conjugated goat anti-mouse and donkey anti-sheep, Alexa 555–conjugated donkey anti-rabbit, and Alexa-647–conjugated goat anti-rabbit and chicken anti-mouse (Life Technologies) were obtained from the indicated suppliers.

Cells were lysed in radioimmunoprecipitation assay buffer (Tris-HCl, pH 7.4, 50 mM; NaCl, 150 mM; NP-40 1%; sodium deoxycholate, 0.5%; sodium dodecyl sulfate, 0.1%; EDTA, 2 mM; sodium fluoride, 50 mM) supplemented with protease and phosphatase inhibitor cocktails (Roche, Mississauga, Canada). Protein concentrations were determined by Bradford protein assay (Bio-Rad, Mississauga, Canada). Loading buffer was added to 30 μg of protein lysate and resolved by SDS–PAGE. The protein was then transferred onto a polyvinylidene difluoride membrane (Millipore, Toronto, Canada) and probed using antibodies for PI4KIIIβ (BD Biosciences 611817; Mississauga, Canada), pan-actin (Cell Signaling Technology 4968), tubulin (1:1000, Cell Signaling Technology 3873; Whitby, Canada), mouse α-FAK (1:200 Thermo Fisher #396500), rabbit α-Phospho-FAK (1:200 ThermoFisher #44-626), as well as anti-rabbit HRP-linked (Cell Signaling Technology catalogue no. 7074). Bands were detected with a MicroChemi chemiluminescent system (DNR Bio-Imaging Systems, Toronto, Canada) and intensities were quantified by densitometry using GelQuant (DNR Bio-Imaging Systems).