rabbit polyclonal anti hcls1  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Selective Azapeptide CD36 Ligand MPE-298 Regulates oxLDL-LOX-1-Mediated Inflammation and Mitochondrial Oxidative Stress in Macrophages

doi: 10.3390/cells14050385

Figure Lengend Snippet: MPE-298 attenuates oxLDL-induced mitochondrial damage in a CD36-dependent manner in primary M1 bone-marrow-derived macrophages (BMDM). ( A ) Western blots of the total and phosphorylated Src (Tyr416), Lyn (Tyr397), and JNK (Thr183/Tyr185) kinases and p66Shc (Ser36) M1-phenotype BMDMs. Differentiated cells from wild-type (WT) and CD36-KO mice were preincubated with or without the LOX-1 inhibitor BI-0115 (5 μM), followed by treatment with MPE-298 (100 nM) or oxLDL (25 μg/mL), or with a combination of MPE-298 and oxLDL. Representative blots of two experiments. The effect of MPE-298 on oxLDL-induced ( B ) mtROS production and ( C ) ΔΨM loss in the absence or presence of BI-0115. The experiments are expressed as the mean ± SEM of two independent experiments each conducted in triplicate. Data were assessed as the fold change relative to vehicle and are presented as the mean ± SEM. Two-way ANOVA test with Tukey’s multiple comparison post-test was performed. * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. vehicle; # p < 0.05 and ### p < 0.001 vs. oxLDL-treated cells.

Article Snippet: Antibodies against early endosome antigen 1 (EEA1), Rab7, Rab11, lysosome-associated membrane protein (Lamp) 1, Rac1/2/3, phospho-Tyr416 and total Src family kinases, phospho-Tyr397 and total Lyn, JNK and phospho-(Thr183/Tyr185) JNK, all were purchased from Cell Signaling (Danvers, MA, USA).

Techniques: Derivative Assay, Western Blot, Comparison

Journal: Life Science Alliance

Article Title: SNX5 promotes antigen presentation in B cells by dual regulation of actin and lysosomal dynamics

doi: 10.26508/lsa.202402917

Figure Lengend Snippet: (A) Representative epifluorescence images of Ctrl and SNX5-silenced B cells incubated with antigen-coated beads for different time points, stained for F-actin (red). Images are presented as z-projections of a stack. White circles indicate bead positions. The scale bar in bright field corresponds to 3 μm. (B) Quantification of the percentage of F-actin fluorescence around the bead. Values were normalized by the initial fluorescence (0 min). Two-way ANOVA with Sidak’s multiple comparison test was conducted for three independent experiments, with n > 25 cells. (C) Representative confocal images of Ctrl and SNX5-silenced B cells seeded onto antigen-coated coverslips for 30 and 60 min and stained for F-actin (red). Non-activated cells were seeded on poly-L-lysine. (D) Quantification of the spreading area of F-actin (μm 2 ) from three independent experiments, with n > 30 cells. Two-way ANOVA with Sidak’s multiple comparison test was performed. * corresponds to the mean with respect to its control. & is the comparison of the mean between siCtrl and siSNX5-B. &&&& < 0.0001. (C, E) Quantification of actin foci in B cells from the experiment shown in (C) at 60 min of activation, for two independent experiments, with n > 25 cells. A t test was conducted. (F) Representative epifluorescence images of Ctrl and SNX5-silenced B cells incubated with antigen-coated beads for 10 and 30 min stained for F-actin (red) and B-cell receptor (green). The stack where the IS is located is shown. (G) Quantification of the B-cell receptor recruitment index to the center of the IS. Two-way ANOVA with Sidak’s multiple comparison test was conducted for two independent experiments, with n > 25 cells. (H) Representative confocal images of Ctrl and SNX5-silenced B cells seeded onto antigen-coated coverslips for 60 min and stained for p-HS1. The stack where the IS is located is shown. Dashed lines indicate cell boundaries (scale bars: 3 μm). (H, I) Quantification of fluorescence intensity of p-HS1 from images in (H). Two-way ANOVA with multiple comparison test under Tukey’s criteria was performed for two independent experiments. * corresponds to the mean with respect to its control. & is the comparison of the mean between siCtrl and siSNX5-B. &&&& < 0.0001.

Article Snippet: The following primary antibodies were used for immunofluorescence: F(ab′) 2 goat anti-mouse immunoglobulin G (IgG) (Jackson ImmunoResearch), rabbit anti-SNX5 (#180520, 1:200; Abcam), rat anti-α-tubulin (#ab6160, 1:500; Abcam), rat anti-(LAMP1) CD107a (#553792, 1:200; BD Biosciences), rabbit anti-OVA (#C6534, 1:500; Sigma-Aldrich), chicken anti-GFP (#ab13970; Abcam), and rabbit anti-pHS1 and HS1 (#4557S and #8714S, 1:200; Cell Signaling).

Techniques: Incubation, Staining, Fluorescence, Comparison, Control, Activation Assay