rabbit anti arp2  (Proteintech)

Journal: bioRxiv

Article Title: High-motility pro-tumorigenic monocytes drive macrophage enrichment in the tumor microenvironment

doi: 10.1101/2024.07.16.603739

Figure Lengend Snippet: ( A ) Illustration cartoon of exogeneous cytokine treatment. Monocytes in 3D collagen matrix were incubated for 6 h with 1 ng/mL of IL-6 or IL-8 and then tracked for motility. ( B ) Speeds and motile fractions of monocytes treated with IL-6, IL-8 or IL-6+IL-8. MDA CM- induced TAMo was used as a positive control. ( C ) Surface expressions of IL-6R, IL-8R and CCR2 on classical human monocytes via flow cytometry. Positivity was gated using fluorescence minus one (FMO) control. ( D ) Classical (71.0%), intermediate (11.0%), and non-classical (10.6%) monocytes were gated via CD14/CD16 surface expressions after pan-monocyte isolation. ( E ) Single-cell atlas of human breast cancer with annotated cell subsets (GSE176078) and IL6R and CCR2 expression (high – blue; low – gray). ( F ) Comparison of IL-6 secretion between high-density MDA-MB-231 tumor cores (to mimic tumor microenvironment) and low-density monocytes or MDA-MB-231 cells embedded in 3D collagen gel at 100 cells/mm 3 . N = 3 biological replicates. Unpaired t test with Welch’s correction was used for statistical analysis (**P≤0.01, *P≤0.05, ns P˃0.05). ( G ) IL-6 ELISA from TAMos under indicated conditions. N=3 biological replicates, n = 3 technical replicates. ( H ) Illustration cartoon of functional IL-6R blockade via tocilizumab. 1 ng/mL exogenous IL-6 and 150 nM tocilizumab were added simultaneously to monocytes and incubated for 6 h at 37°C, 5% CO2 before tracking. ( I ) Speeds and motile fractions of monocytes under indicated treatments. ( J ) Top 10 downregulated gene sets in TAMos when IL-6/IL-6R interaction was inhibited. Cell migration related biological processes were labeled red. ( K ) Representative lattice light sheet microscopy images recording active dynamics of cell dendritic protrusions. Scale bar, 5 μm. ( L ) Illustration cartoon showing that IL-6 signaling pathway was inhibited via targeting at downstream transcription factor STAT3 and actin nucleator Arp2/3 complex. ( M-N ) Speeds and motile fractions of monocytes following treatment of 10 μM STAT3 inhibitor S3I-201 or 50 μM Arp2/3 inhibitor CK-666. ( O-P ) Representative western blot results for Arp2 and pMLC2 expression in monocytes treated as indicated. ( Q ) Speeds and motile fractions of TAMos with or without inhibiting myosin II activity using 50 μM blebbistatin. For migration assays, N = 2 donors, n = 50 for a total of 100 individual cells were tracked for each condition in all experiments. Speeds and motile fractions (N = 2 donors, n = 5 technical replicates) are plotted as mean + standard deviation. Non-parametric Mann-Whitney test was used for statistical analysis comparing 2 groups (****P≤0.0001, **P≤0.01, ns P˃0.05). Kruskal-Wallis ANOVA with Dunn’s test was used for statistical analysis comparing groups with control (****P≤0.0001, **P≤0.001, *P≤0.05, ns P˃0.05).

Article Snippet: Primary and secondary antibodies were used as follows: α-Tubulin (11H10) Rabbit mAb (CST, 2125), Arp2 (D85D5) Rabbit mAb (CST, 5614), phospho-myosin light chain 2 (Ser19) antibody (CST, 3671), Anti-Rabbit IgG HRP-linked Antibody (CST, 7074), IL6 (D5W4V) XP rabbit mAb (CST, 12912).

Techniques: Incubation, Positive Control, Flow Cytometry, Fluorescence, Control, Isolation, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Functional Assay, Migration, Labeling, Microscopy, Western Blot, Activity Assay, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: A protein kinase A-regulated centrosomal actin pool sets the threshold for T cell polarization

doi: 10.1101/2024.06.03.597129

Figure Lengend Snippet: a- Example of distribution of F-Actin and Arp2 in representative cells in control conditions (upper line), after CXCL12 stimulation (middle line) or after H89 pretreatment (lower line). Scale bar = 10µm. b- After CXCL12 stimulation, the accumulations of F-Actin and Arp2/3 at the centrosome are both decreased while they are enhanced after preincubation with the PKA inhibitor H89. The same cells have been quantified for the 2 labeling. Mean ± SE of 4 independent experiments. 38-165 cells/condition/experiment.

Article Snippet: The following primary antibodies were used for immunofluorescence: mouse anti-pericentrin (Abcam, #28144, 1/1000), rabbit anti-pericentrin (Abcam, #4448, 1/2000), rabbit anti-Arp2 (Abcam, #47654, 1/200), anti-AKAP450 (Becton Dickinson 611518, 1/50), Mouse anti CXCR4 human (R et D Systems, #MAB172, 1/200).

Techniques: Labeling