Ship 1

After washing the cells three times with PBS, they were lysed on ice in radioimmunoprecipitation analysis buffer (RIPA; Beyotime, China Institute of Biotechnology, 1% phenylmethylsulfonyl fluoride (PMSF), and 1% NaF for 30 min. The samples were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected. Next, the proteins were separated on SDS–PAGE gels and transferred to a PVDF membrane (Merck Millipore, Burlington, Massachusetts, USA). The membrane was incubated with primary antibodies against β-actin (1:1000, Cell Signaling Technology), TIGIT (1:1000, Cell Signaling Technology), SHIP-1 (1:1000, Cell Signaling Technology), ERK (1:1000, Cell Signaling Technology), p-ERK (1:1000, Cell Signaling Technology), p-IκBα (1:1000, Cell Signaling Technology), p-NF-κBP65 (1:1000, Cell Signaling Technology) overnight at 4 °C, and then incubated with the appropriate secondary antibody. Image J software (National Institutes of Health) was used to analyse relative protein levels, and β-actin was used as an endogenous control.

Purified splenic B cells (2 × 10⁶) were activated at 37°C with sAg for 5, 10 and 30 min. Cell lysates were obtained using a mixed RIPA buffer, as previously described.²⁴ Lysates were run through SDS‐PAGE and western blotting. Abs from Cell Signaling Technology: BTK (8547S), pAKT (4060L), AKT (9272S), SHIP‐1 (2728S), pFOXO1 (9461S), FOXO1 (2880S), pS6 (4856S), S6 (2217S), pPI3K (4228S), PI3K (4292S), pMST1 (3681S), MST1 (PA5‐22015), pmTOR (5536S), mTOR (2983S), pEZRIN (3726S), STAT1 (14994S), P65 (4764S), pIKKB (2697S), IKKB (8943S), and anti‐human‐CCR2 (12199S). From Abcam: STAT5 (ab194898). From Santa Cruz Biotechnology: WASP (sc‐13139). Loading controls: anti‐mouse β‐actin (60008‐1‐IG‐10, Proteintech), anti‐human GAPDH (5174S, Cell Signaling Technology). Western blotting imaging was performed using the ChemiDoc™XRS + imaging systems (Bio‐Rad).

WT BMDCs were collected from cultures on day 6, treated with curdlan or dZym (50 µg/mL), and then harvested at indicated time points. Cells were lysed and the lysates were precleared with protein A/G beads (G-Bioscience) at 4°C for 2 h. After removing the beads, the lysates were incubated with anti-FcRγ Ab (06-727, Millipore) at 4°C for 16 h and then protein A/G beads were added for precipitating Ab-conjugated proteins. After 2 h, the precipitated proteins were resolved by SDS-PAGE and then evaluated by Western blot using anti-SHP-1 (#3759), -SHP-2 (#3397), -SHIP-1 (#2728, all from Cell Signaling), -FcRγ and -PTEN (04-409, Millipore), and -actin Abs. Quantification was determined by densitometry using ImageJ software.