Journal: PLoS ONE

Article Title: Radotinib Induces Apoptosis of CD11b + Cells Differentiated from Acute Myeloid Leukemia Cells

doi: 10.1371/journal.pone.0129853

Figure Lengend Snippet: (A) Radotinib decreased LYN activity in NB4 cells but increased it in HL60 cells. Cells were stimulated with 0, 1, and 5 μM radotinib or dasatinib for 48 h. After immunoprecipitation of LYN, immunoprecipitate pellets were subjected to immunoblotting with antibodies against phosphor-SFK (Y416), c-Raf, ERK, and LYN in NB4 and HL60 cells. (B) The relative band density is shown. Data are presented as the mean ± SEM. Statistically significant differences from the DMSO-treated control are given as follows. **: P < 0.01; ***: P < 0.001. Rd, radotinib; Das, dasatinib.

Article Snippet: Antibodies against ERK, phospho-SFK Y416, c-Raf, cleaved caspase-3, and anti-rabbit IgG-HRP were obtained from Cell Signaling Technology (Beverly, MA).

Techniques: Activity Assay, Immunoprecipitation, Western Blot, Control

Journal: The Journal of Experimental Medicine

Article Title: Fyn kinase regulates misfolded α-synuclein uptake and NLRP3 inflammasome activation in microglia

doi: 10.1084/jem.20182191

Figure Lengend Snippet: Fyn and CD36 contribute to aggregated αSyn uptake into microglia . (A) Immunoblot analysis of aggregated αSyn-treated WT and Fyn −/− microglial lysates reveals a rapid induction of SFK activity in WT, but not Fyn −/− , microglia. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). Con, control. (B) ICC reveals rapid increase in p-Y416 SFK levels in αSyn-treated Iba-1–positive WT microglial cells. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4). (C) IHC analysis to show microglial Fyn activation in the AAV-SYN model at 30 d. Schematic of AAV injection on the side. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4 mice per group). (D) Upon its application to microglial cells, αSyn associates with TLR2 and CD36, as evidenced by coIP analysis. Upon αSyn treatment, Fyn associates with CD36, but not TLR2. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). IP, immunoprecipitation. (E) Immunoprecipitation of FLAG-tagged human CD36 in αSyn-treated HEK cells shows a transient interaction of CD36 and αSyn. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). IB, immunoblotting. (F) Immunoblot for p-Y416 SFK reveals that Fyn activation occurs downstream of CD36. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 3). (G) Immunoblot analysis showing diminished αSyn uptake by CD36 −/− BMDMs. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). (H) Immunoblot analysis also reveals that reduced αSyn was taken up by Fyn −/− microglia. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 5). (I and J) ICC for human αSyn revealed diminished uptake of the protein in Fyn-deficient microglia. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 5). Scale bars, 15 µm. (K) Whole-cell lysate analysis from αSyn-treated, WT-transfected, and activation loop–deficient (Y417A) Fyn-FLAG–transfected cells showed diminished αSyn uptake in inactive Fyn mutant–expressing cells. Error bars represent mean ± SEM. One-way ANOVA followed by Tukey’s post hoc test ( n = 3). Asterisks indicate the level of statistical significance: ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.

Article Snippet: Antibodies against the rabbit p-Y416 SFK and native p65 were purchased from Cell Signaling.

Techniques: Western Blot, Activity Assay, Control, Two Tailed Test, Activation Assay, Injection, Immunoprecipitation, Transfection, Mutagenesis, Expressing

Journal: The Journal of Experimental Medicine

Article Title: Fyn kinase regulates misfolded α-synuclein uptake and NLRP3 inflammasome activation in microglia

doi: 10.1084/jem.20182191

Figure Lengend Snippet: Microglial Fyn activation in human PD brains. (A) Significantly increased Fyn expression and activation were observed in AAV-SYN–injected nigral lysates, as assessed by immunoblots. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 4 mice per group). (B) IHC analysis revealed Fyn induction in Iba-1–positive microglia in AAV-SYN–injected nigral brain sections. Scale bars, 30 µm. (C) Significant increase in Fyn expression and activation in PD ventral midbrain lysates, when compared with age-matched control lysates. Representative immunoblot is shown. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 12). (D) IHC analysis of PD brain sections revealed strongly increased p-Y416 SFK expression within Iba-positive microglia when compared with age-matched non-PD brain sections. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 6). Scale bars, 15 µm. (E) IHC shows increased microglial Fyn expression in PD ventral midbrain sections. Scale bars, 15 µm. Error bars represent mean ± SEM. Unpaired two-tailed t test ( n = 6). Asterisks indicate the level of statistical significance: *, P ≤ 0.05; ***, P ≤ 0.001.

Article Snippet: Antibodies against the rabbit p-Y416 SFK and native p65 were purchased from Cell Signaling.

Techniques: Activation Assay, Expressing, Injection, Western Blot, Two Tailed Test, Control

Journal: The Journal of Biological Chemistry

Article Title: A new paradigm for regulation of protein phosphatase 2A function via Src and Fyn kinase–mediated tyrosine phosphorylation

doi: 10.1016/j.jbc.2022.102248

Figure Lengend Snippet: Distinct effects of pervanadate, Scr CA , and Fyn CA on PP2Ac WT, Y127F, Y284F, and Y127/284F tyrosine phosphorylation. Lysates and HA-immunoprecipitates were prepared from control and pervanadate-treated (+PV) Cos-7 cells ( A and B ), Src CA -transfected Cos-7 cells ( C and D ), and GFP-Fyn CA -transfected N2a cells ( E ) that were cotransfected with plasmids encoding HA3-tagged PP2Ac wildtype (WT), Y127F (Y127F), Y284F (Y284F), Y127/284F (F/F), or the empty vector (EV). Representative Western blots with the indicated antibodies are shown in ( A ), ( C ), and ( E ). Note: the pSFK antibody recognizes Src only when phosphorylated at Y416. B , relative levels of pY signal in HA3-PP2Ac immunoprecipitates from ( A ). Data (mean ± SEM from n = 3 separate experiments) were appraised using one-way ANOVA (F = 236; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared with WT; #### p < 0.0001, # p < 0.05. D , relative levels of pY signal in HA3-PP2Ac immunoprecipitates from ( C ). Data (mean ± SEM from n = 3–4 separate experiments) were appraised using one-way ANOVA (F = 2067; p < 0.0001) with Tukey’s post hoc multiple comparisons test. ∗∗∗∗ p < 0.0001, compared with WT; #### p < 0.0001. ( E ) similar results were observed in three separate experiments.

Article Snippet: Antibodies used in this study included mouse anti-HA (clone 16B12, Covance); rabbit anti-HA (clone C29F4, Cell Signaling Technology); rabbit anti-GFP (clone D5.1, Cell Signaling Technology); mouse anti-Bα (clone 2G9, Merck Millipore); mouse anti-PP2Ac α/β (BD Transduction); goat anti-pY307 PP2Ac (Santa Cruz Biotechnology, Inc; denoted SC); rabbit anti-phospho-SFK (Y416) (clone 100F9, Cell Signaling Technology); rabbit anti-phospho-p44/42 MAPK (clone D13.14.4E, Cell Signaling Technology); mouse anti-p44/42 MAPK (ERK1/2) (L34F12, Cell Signaling Technology); rabbit anti-phospho-GSK-3β (Ser 9) (Cell Signaling Technology) and mouse anti-GSK3β (clone 3D10, Cell Signaling Technology); rabbit anti-Src (clone 32G6, Cell Signaling Technology); mouse anti-Src (clone GD11, Merck Millipore); rabbit anti-Fyn (clone EPR5500, Merck Millipore); mouse anti-Fyn (BD Transduction and Cell Signaling Technology); mouse and rabbit anti-p-Tyr (P-Tyr-100 and P-Tyr-1000, Cell Signaling Technology); rabbit anti-Tau (rPeptide), mouse anti-pSer202 Tau (CP13) ( ) (a gift from Peter Davies).

Techniques: Phospho-proteomics, Control, Transfection, Plasmid Preparation, Western Blot