2 mm glutamax

Whole intestines were removed from E17 chick embryos into DMEM-F12 medium (Sigma- Aldrich Company, Ayrshire. UK). Individual Intestines were washed and segmented into duodenum (pancreatic loop), cecum (two ceca were disconnected from the intestines at the ileo-cecal junction) and colon. Each segment was then added to a pool of identical segments. The pancreas was removed from duodenal loops, and loop segments were split lengthwise and sliced using a sterile scalpel blade. Slices were forced thru a stainless steel mesh (55mm diameter 100 micron opening, Sigma-Aldrich, Rehovot IL). The resulting fragments were collected, washed and suspended in complete DMEM-F12 medium containing: penicillin 100 U/ml, streptomycin 0.1 mg/ml, nystatin 12.5 U/ml, sodium pyruvate 0.11 mg/ml (all from Biological Industries, Beit Haemek, Israel), Glutamax™ 2 mM (Thermo-Fisher Scientific, Carlsbad, CA, USA), and BD™ Mito-serum Extender (BD Biosciences, Bedford, MA, USA. The resulting suspension was seeded into six well plates (Nunc), previously thinly coated with BD Matrigel matrix (Erembodegem, Belgium). The same procedure was applied to segments derived from ceca and colon. Plates were incubated at 37.5°C, 7.5% CO₂. Under these conditions, epithelial monolayers develop within 48h.

The C57BL/6 mice were maintained and bred in in the Animal and Plant Care Facility of The Hong Kong University of Science and Technology (HKUST). Primary cortical cultures were performed following standard protocols as described (Hui et al., 2016 (link)). Cervical dislocation was performed to kill the pregnant female C57BL/6 mice, followed by isolation of embryonic mice. Isolated cerebral cortices from E16.5 or P5 C57BL/6 mice were dissociated into single cells by digestion with 0.25% trypsin-EDTA (Thermo Fisher Scientific) and gentle trituration. After neutralization with DMEM containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific), cells were suspended in Neurobasal medium supplemented with B27 and 2 mm GlutaMAX (Thermo Fisher Scientific), and then seeded on glass coverslips or dishes coated with poly-L-lysine (Sigma). Half of the medium was replaced with fresh every 5 d.

Embryonic cortical neurons were isolated by standard procedures. Embryonic day 16.5 (E16.5) embryonic cerebral cortices were treated with 0.25% trypsin-EDTA (ThermoFisher Scientific) and dissociated into single cells by gentle trituration. Cells were suspended in Neurobasal medium supplemented with B27 and 2 mm GlutaMAX (ThermoFisher Scientific), then they were plated on coverslips or dishes coated with poly-l-lysine (0.05 mg/mL; ThermoFisher Scientific). All cultures were grown for a minimum of 14 d in vitro before harvest. Genotyping was performed after plating the neurons.

HEK293 cells were grown in Dulbecco's modified eagle medium (DMEM) supplemented with 10% (v/v) Fetal Bovine serum (GE Healthcare, Little Chalfont, UK), 2 mm glutaMAX (ThermoFisher Scientific, Waltham, MA, USA), 100U·mL⁻¹ Penicillin and 100 μg·mL⁻¹ Streptomycin (ThermoFisher Scientific). Anti‐GST, Anti‐FLAG M2 antibody, HA antibody and agarose resins were purchased from Sigma (St. Louis, MO, USA). Glutathione sepharose 4B beads were from GE healthcare. Anti‐phosphotyrosine antibody (4G10) was from Millipore (Billerica, MA, USA), PKD anti‐pSer‐744/748 antibody, anti‐PKCδ antibody, secondary HRP‐linked goat anti‐Rabbit and Horse, anti‐Mouse antibodies were from Cell Signaling Technologies (Beverly, MA, USA). An in‐house site‐specific phospho‐Tyr antibody targeting pTyr in the P + 1 loop (CPApYLAPEV), which is cross‐reactive between PKD isoforms due to 100% homology of the epitope is described previously 36. Phorbol 12,13‐dibutyrate (PDB), ATP, STI‐571, PP2, CRT 0066101, CID 755673 and Hydrogen peroxide 30% (v/v) were from Sigma, Polyethyleneimine (PEI) was from Polysciences Inc. (Warrington, PA, USA).