Fitc gelatin

In brief, coverslips were incubated with 0.15% glutaraldehyde/phosphate-buffered saline (PBS) for 10 min followed by PBS washes. A total of 100 μL of 1:9 FITC–gelatin (Invitrogen): 0.2% porcine gelatin was added. Coverslips were then washed in PBS and incubated 15 min in 5 mg mL⁻¹ NaBH₄. Coverslips were rinsed in PBS and incubated at 37 °C in 10% FBS/DMEM for 2 h. MDA-MB-231 cells were seeded on coverslips with or without WJ460. Cells were incubated for 12 h and processed for immunofluorescence.

Commercial FITC-gelatin was acquired from Invitrogen. The preparation of rhodamine B conjugated-alginate was as follows. First, rhodamine B (20 mg; Invitrogen) added to 2 mL DPBS; then, 1-ethyl-3-(3 (dimethylamino) propyl) carbodiimide (EDC; 40 mg; Thermo Fisher Scientific) and 1-hydroxy-2-line 5-pyrrolidine dione (NHS; 1.75 mg; Thermo Fisher Scientific) were added; the sample was incubated at room temperature (25 ± 1 °C) for 30 min. Ethylenediamine was subsequently added and the sample incubated for 12 h. After dialysis and freeze-drying, rhodamine B-ethylenediamine powder was dissolved in 1% alginate solution (2 mL) and adding EDC (10 mg). The solution was oscillated overnight at room temperature, dialyzed, and freeze-dried to obtain rhodamine B-alginate powder.

CCA cells were seeded onto FITC-gelatin (Thermo Scientific) substrates in a 12-well plate. After 24 h of culture, cells were fixed with methanol and TBK1 was stained through immunefluorescence. Finally, images were acquired using a fluorescence microscope.

For Western blotting analysis, primary B cells, macrophages, or B cell lines were lysed in RIPA lysis buffer (MilliporeSigma, R0278) containing protease inhibitors (Thermo Fisher Scientific, A32963) and phosphatase inhibitors (Thermo Fisher Scientific, A32957). Antibodies used for blotting were from Cell Signaling Technology including anti-phospho-473-Akt (catalog 4060); anti-Akt (catalog 2920); anti-phospho-p44/42 MAPK (ERK1/2) Thr202/204 (catalog 9101); and anti-p44/42 MAPK (ERK1/2) (catalog 4695). Antibodies used for immunostaining included anti-Talin (MilliporeSigma, T3287), anti-Vinculin (Thermo Fisher Scientific, 700062), anti-HS-1 (Cell Signaling Technologies, 3890), and anti-ARPC1B (MilliporeSigma, HPA004832). Alexa-488 or -647 Phalloidin were from Thermo Fisher Scientific. siRNA for ARPC1B was from Horizon Inspired Cell Solutions (catalog L-012082-00-0005). Lipopolysaccharide was from MilliporeSigma (catalog L6511). FITC-gelatin was from Thermo Fisher Scientific (catalog G13187). Fluo-8 AM was from Abcam (catalog ab142773). SYK inhibitors BAY 61-3606 (Selleck) or R406 (AdooQ Bioscience) were used at 500 nM.

FITC-gelatin (Thermo Fisher Scientific Waltham, MA) was used to coat acid-pretreated 22 mm × 22 mm coverslips. Cells (1 × 10³) were seeded on the FITC-gelatin-coated coverslips. The clear zone of FITC-gelatin was determined using an Olympus DP-80 microscope image system (Olympus, Tokyo, Japan). Images were quantitated by ImageJ software.