Figure 1.

sGSN inhibits DNGR-1 binding to F-actin

(A–C) Serial (2-fold) dilutions (wedge) of in vitro polymerized F-actin (top concentration 0.2 μM) or no F-actin (PBS; arrows) were spotted onto a membrane. DNGR-1 ECD (5 μg/mL) binding to the dots was detected following pre-treatment of the membrane with (A) the indicated doses of FCS, (B) ABP-depleted or mock-depleted FCS, and (C) sGSN or cofilin (both at 10 μg/mL).

(D, and E) Flow cytometric analysis of bead-bound F-actin treated or not with (D) 10 μg/mL sGSN or (E) 10 μg/mL sGSN in the presence or absence of Ca²⁺ before staining with DNGR-1 ECD, anti-GSN, or anti-actin antibodies. Numbers above graphs represent mean fluorescence intensity for each of the three samples.

(F) Generation of sGsn^−/− mice using CRISPR/Cas9 technology and sgRNA pairs that target the signal peptide sequence. Table shows different enzymatically modified (em1–8) mutant alleles generated and their predicted protein sequence.

(G) Serum (top panel) and spleen lysates (bottom panel) from intercrossed littermate mutant mice genotyped for the indicated alleles were immunoblotted for the indicated proteins. WT indicates mice that after genotyping were deemed sGsn^+/+. Homozygous line em2 was selected for further characterization and is henceforth referred to as sGsn^−/− mice.

(H) Dot blot analysis of DNGR-1 ECD binding to immobilized F-actin, pre-treated or not with FCS or 10% mouse serum from WT or sGsn-deficient mice. #1 and #2 represent serum from individual mice.

Data are representative of (E) two, (B, C, and H) three, and (A and D) six independent experiments. See also Figure S1.