Journal: bioRxiv
Article Title: Steady-state phosphorylation of SHIP1 by Lyn restricts macrophage activation in the absence of a phagocytic synapse
doi: 10.1101/2025.04.16.648985
Figure Lengend Snippet: (A-D) Immunoblots and quantifications of lysates from IFN-γ-primed Lyn +/+ and Lyn KO BMDMs probed for phosphorylated (A) Syk Y352 , (B) Erk1/2 T202/Y204 , (C) Akt S473 , and (D) SHIP1 Y1020 . β-Actin is a visual loading control, and quantifications were normalized to total protein content in the gel lane. Data are shown relative to the signal in Lyn +/+ . n=10-15 for Lyn +/+ and 6-7 for Lyn KO . Error bars: SEM. Sig. nonparametric, unpaired t with Kolmogorov-Smirnov test, *P=0.0152, **P≥0.007, ***P=0.0006 and ****P≤0.0001. (E) Representative images of PIP 3 (yellow) and nuclear staining (cyan) in IFN-γ -primed BMDMs. (F) Quantification of the geometric mean fluorescence intensity (gMFI) of PIP 3 staining in Lyn KO relative to Lyn +/+ BMDMs. Data points reflect the mean and SEM of 3-4 independent wells from 4 biological replicates. Sig. nonparametric, unpaired t test with Kolmogorov-Smirnov test, *** P<0.0006.
Article Snippet: Antibodies were obtained from Cell Signaling Technologies (rabbit anti-pSyk Y352 65E4 ID/catalog # (#)2717, rabbit anti-pPLCγ Y1217 #3871, rabbit anti-pSHIP1 Y1020 #3941, rabbit anti-pSHP-1 Y564 D11G5 #8849, rabbit anti-pErk1/2 T202/Y204 D13.14.4e #4370, rabbit anti-pAkt S473 193H12 #4058, rabbit anti-pPI3K p85(Y458)/p55(Y199) #4228, mouse anti-β-Actin 8H10D10 #3700), Abcam (Cambridge, United Kingdom) (mouse anti-LynA+B Lyn01 ab1890), Santa Cruz (Dallas, TX, USA) (rabbit anti-LynA+B 44 #sc-15, goat anti-Hck M-28 #sc-1428), and LICOR (donkey anti-mouse IgG 800CW #925-32212, donkey anti-rabbit IgG 800CW #925-32213, donkey anti-mouse IgG 680LT #925-68022, donkey anti-goat IgG 680LT #926-32214 ) .
Techniques: Western Blot, Control, Staining, Fluorescence