Bp358

For polysaccharides (GAG or curdlan [β-glucan; C7821, Sigma]) or flagellin (tlrl-epstfla, Invivogen) transfection, BMDMs were incubated with 1 μg/mL of Pam3CSK4 (tlrl-pms, Invivogen) for 4 h, washed, and incubated for 1 h prior to transfection in HBSS/Modified (HyClone, SH30031.02). Then 20 μg/mL GAG, Ac-GAG, d-GAG or curdlan were resuspended in HCl 0.01 N or 2 μg/mL flagellin was resuspended in LAL water (Invivogen) and mixed with DOTAP (Roche, 11202375001) as per the manufacturer’s protocol (vol:vol ratio).
For poly(dA:dT) (tlrl-patn, Invivogen) transfection, the BMDMs were incubated with 1 μg/mL of Pam3CSK4 for 4 h, washed, and were incubated 1 h prior to transfection in Opti-MEM (31985–070, Thermo Fisher Scientific). One μg/mL of poly(dA:dT) was mixed with Xfect (631318, Takara) as per the manufacturer’s protocol. The BMDMs were stimulated with mixed solutions for 3 h before cell lysate collection.
For inhibition of the proteasome or salt treatment during GAG transfection, the medium was complemented 10 min prior to transfection with 30 μM of MG132 (M8699, Sigma Aldrich) or 200 mM of NaCl (BP358, Fisher).
To measure poly-ubiquitination, the cells were washed with PBS and harvested with RIPA buffer complemented with 10 μM of N-Ethylmaleimide (NEM).

For identification of proteins interacting with GAG, the BMDMs were lysed in NP40 buffer (50 mM HEPES, 150 mM NaCl, 1% NP40, 50 mM EDTA, pH 7.4 ± 0.2) and centrifuged for 10 min at 10,000 × g to remove cell debris. Cell lysate and GAGs (GAG, Ac-GAG, d-GAG) or β-glucan were incubated for 2 h with rotation at room temperature with or without 1.2 M NaCl (BP358, Fisher). The samples were washed 6 times with NP40 buffer after 10 min of 10,000 × g centrifugation at 4°C. After washes, polysaccharides pulled down were eluted in sample buffer and used for immunoblotting analysis or mass spectrometry analysis on the Q-Exactive mass spectrometer as describe previously^{17 (link)}.