Journal: Cell Reports

Article Title: CDK8 Fine-Tunes IL-6 Transcriptional Activities by Limiting STAT3 Resident Time at the Gene Loci

doi: 10.1016/j.celrep.2020.108545

Figure Lengend Snippet:

Article Snippet: Finally, cells were pooled and stained with anti-CD3 BV510 (Biolegend, #300448), anti-CD4 PE (Biolegend, #357404), anti-CD8 AF700 (Biolegend, #300920), anti-pSTAT1-Y701 AF647 (Cell Signaling, #8009S), anti-pSTAT1-S727 AF488 (Biolegend, #686410), anti-pSTAT3-Y705 AF488 (Biolegend, #651006) and anti-pSTAT3-S727 AF647 (Biolegend, #698913), anti-pSTAT4-Y693 AF488 (BD Biosciences, #558136), anti-pSTAT5-Y694 AF647 (Cell Signaling, #9365S), anti-pSTAT6-Y641 AF488 (BD Biosciences, #612600), anti-pERK-T202/Y204 AF488 (eBiosciences, #53-9109-41), anti-pAKT-S473 AF488 (Cell Signaling, #4071S), anti-pAKT-T308 AF647 (Cell Signaling, #48646S), anti-pP90RSK-S380 AF488 (Cell Signaling, #13588S), anti-pS6R-S240/S244 AF488 (Cell Signaling, #5018S), anti-pS6R-S235/S236 AF647 (Cell Signaling, #4851S), anti-pZAP70-Y319/pSYK-Y352 AF647 (Cell Signaling, #82975S), anti-pCREB-S133 AF488 (Cell Signaling, #9187S), anti-pHIS3-S10 AF647 (Cell Signaling, #9716S), anti-pGSK3β-S9 AF647 (Cell Signaling, #14332S),anti-pCFOS-S32 AF647 (Cell Signaling, #8677S), anti-IRF1 AF647 (Cell Signaling, #14105S), anti-IRF4 AF647 (Biolegend, #646408), anti-IRF7 AF647 (Biolegend, #656007), anti-GATA3 AF488 (Biolegend, #653807), anti-TBET AF647 (Biolegend, #644803), anti-HIF1α AF488 (Biolegend, #359707), anti-MYC AF488 (Cell Signaling, #12855S), anti-O-GlcNAC AF647 (NOVUS Biologicals, #NB300-524AF647), anti-STAT3 APC (BD Biosciences, #560392) and anti-PLCγ1 AF647 (BD Biosciences, #557883).

Techniques: Purification, Recombinant, Phospho-proteomics, Software

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-C) Representative confocal micrographs showing a single z slice (∼280 nm) of IFN-γ-primed Csk AS BMDMs stained for total Lyn (yellow) and pSyk Y352 (cyan) after 3 min treatments with (A) medium only (untreated), (B) 3-IB-PP1, or (C) depleted zy-mosan (zym dep ). Fluorescence in the DAPI channel shows an internalized zym dep (magenta). Scale bars: 5 μm (D) Interaction area for pSyk Y352 clusters formed spontaneously during 3-IB-PP1 treatment and semi-contiguous interaction area per phagocytosed zym dep particle. Points are single-punctum values combined from 5 cells, shown with standard error of the mean (SEM). Significance (Sig.) in all panels assessed via nonparametric t test and Kolmogorov-Smirnov test: *** P = 0.0001. Inset: view of the lower range of 3-IB-PP1-induced pSyk clustering, showing the preponderance of nanoclusters within the distribution.

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: Staining, Fluorescence

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: Densitometry quantification of protein species in immunoblotted lysates from IFN-γ-primed Csk AS BMDMs after 5 min treatment with 3-IB-PP1 or zym dep probed by immunoblot for (A) pSyk Y352 , (B) pPLCγ2 Y1217 , (C) pPI3K p85/p55 (D) immunoprecipitated Ras-GTP, (E) pErk1/2 Y202/T204 , and (F) pAkt S473 . Error: SEM; n=9-14 for 3-IB-PP1 and 11-15 for zym dep . Sig. assessed via non-parametric t test and Kolmogorov-Smirnov test: * P = 0.0216, *** P = 0.0007, ** P < 0.0001.

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, Immunoprecipitation

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

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: Immunoblots and quantifications of (A-B) pSyk Y352 , (C-D) pErk1/2 T202/Y204 , (E-F) pAkt S473 , and (G-H) pSHIP1 Y1020 in IFN-γ-primed Csk AS (Lyn +/+ ) or Csk AS Lyn KO (Lyn KO ) BMDMs treated with (A, C, E, G) 3-IB-PP1 or (B, D, F, H) zym dep , corrected for total protein in each gel lane and shown relative to t=0 of Lyn +/+ ; n=10-11 for Lyn +/+ , 4-7 for Lyn KO . Error bars: SEM. Sig. via two-way ANOVA with Sidak’s multiple comparisons test: * P = 0.0143, ** P = 0.0041, **** P<0.0001.

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

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: Immunoblots and quantification of (A) pSHIP1 Y1020 , (B) pAkt S473 , (C) pSyk Y352 , and (D) pErk T202/Y204 in CskAS (Hck +/+ Fgr +/+ ) and Csk AS Hck KO Fgr KO (Hck KO Fgr KO ) BMDMs, corrected for the total protein in each lane and shown relative to t=0 of Hck +/+ Fgr +/+ . Error bars: SEM, n=10-11 for Hck +/+ Fgr +/+ and n=4 for Hck KO Fgr KO . Sig. 2-way ANOVA with Sidak’s multiple comparisons test: * P = 0.0499.

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

Journal: Inflammation

Article Title: SYK Activation Enhances Dendritic Cell Functions in Spontaneous Rheumatoid Arthritis

doi: 10.1007/s10753-025-02405-2

Figure Lengend Snippet: SYK enhances DC functions. (a) Western blot analysis of WT and SYK S544Y BMDC SYK phosphorylation (Tyr525/526), total SYK expression, JNK1 (T183/Y185) phosphorylation, total JNK1 expression, ERK1 phosphorylation (T203/Y205) and total ERK1 expression treated or not treated with R406 SYK inhibitor. ( n = 3) (b) MTS test of WT and SYK S544Y BMDCs in 24 and 48 h ( n = 3). (c) Percentage of CD11C + cells in BMDC derived from WT and SYK S544Y mice ( n ≥ 5). (d) Percentage of CD40 + , CD86 + , MHCII + cells of total CD11C + in BMDC derived from WT and SYK S544Y mice ( n ≥ 5). (e-f) Percentage and representative plots of mature FITC + BMDCs incubated at 4℃ (f) and 37 ℃ (g) for 1 h. ( n = 5). (g-h) Percentage and representative plots of immature FITC + BMDCs incubated in 4℃ (f) and 37 ℃ (g) for 1 h ( n = 5). (i) Proliferation of OT-II CD8 T cells cocultured with WT or SYK S544Y BMDCs in various ratios ( n = 3). (j) Representative plots show the proportion of proliferative CD8 T cells cocultured with WT or SYK S544Y BMDCs in 20:1, 10:1, and 5:1 ratios. (k) p-SYK expression in WT and SYK S544Y BMDCs after DC-T cell interaction by FACS ( n = 5). In all panels, NS was not significant, ** p < 0.01, *** p < 0.001 and **** p < 0.0001. A p-value less than 0.05 was considered significant

Article Snippet: For intracellular staining, anti-pSYK (Tyr525/526) (Cell Signaling Technology, 4349 S), diluted in Perm/Wash Buffer (BD Biosciences), were applied to cells and allowed to incubate for 30 min. After the staining steps, cells were washed twice with Perm/Wash Buffer and once with FACS buffer.

Techniques: Western Blot, Phospho-proteomics, Expressing, Derivative Assay, Incubation

Journal: Redox Biology

Article Title: ROS via BTK-p300-STAT1-PPARγ signaling activation mediates cholesterol crystals-induced CD36 expression and foam cell formation

doi: 10.1016/j.redox.2016.12.005

Figure Lengend Snippet: CC induces p300 activation in BTK-dependent manner. A. Equal amounts of protein from control and the indicated time periods of CC (40 μg/ml)-treated cells were immunoprecipitated with anti-p300 antibodies or IgG and the immunocomplexes were analyzed by Western blotting with anti-PY20 or anti-phosphoserine/threonine antibodies followed by normalization for p300. B. All the conditions were same as in panel A except that the cell extracts were analyzed by Western blotting for pBTK, pPyk2, pSrc and pSyk levels using their phospho-specific antibodies and normalized for their total levels. C. Cells were transfected with the indicated ASO, quiesced, treated with and without CC and analyzed for CD36 expression, oxLDL uptake or foam cell formation as described in , panel C. The blot was reprobed for BTK, Pyk2 and β-tubulin levels to show the effects of the ASOs on their target and off target molecules levels. D. Cells were transfected with control or BTK ASO, quiesced, treated with and without CC for 1 h and cell extracts were prepared. Equal amount of protein from control and each treatment were immunoprecipitated with anti-p300 or anti-STAT1 antibodies and the immunocomplexes were analyzed by Western blotting for anti-PY20, anti-STAT1, anti-Ac-Lys or anti-PPARγ antibodies and normalized for p300 or STAT1. An equal amount of protein from control and each treatment was also assayed for p300 acetyltransferase activity. *p<0.01 vs control ASO; **p<0.01 vs control ASO+CC.

Article Snippet: Anti-pBTK (5082), anti-pPyk2 (3291), anti-pSrc (2101), anti-pSTAT1 (9177), anti-pSTAT2 (4441), anti-pSTAT3 (9133), anti-pSTAT4 (5267), anti-pSTAT5 (9351), anti-pSTAT6 (9364), and anti-pSyk (2715) antibodies were bought from Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Control, Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay