Anti arp2

Manufactured by Cell Signaling Technology

Anti-ARP2 is a primary antibody that recognizes the ARP2 (Actin-Related Protein 2) subunit of the ARP2/3 complex. The ARP2/3 complex plays a crucial role in the nucleation and organization of actin filaments, which are essential for various cellular processes such as cell motility, vesicle trafficking, and cytoskeletal organization.

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3 protocols using anti arp2

Arp2/3 complex crosslinking dynamics

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Human Arp2/3 complex carrying mutations L199C in Arp2 and L117C in Arp3 (0.25 μM) was incubated with either 2.5 μM WCA, 1.25 µM CapZ-CA (mutant C206A), 1.25 μM pre-purified CapZ-CA/actin, or 5 µM Arpin CA for 30 min at RT in KMEI buffer supplemented with 0.2 mM ATP. BMOE (ThermoFisher Scientific), prepared fresh in dimethyl sulfoxide, was added to a final concentration of 16 μM. Reactions were performed at 21°C and quenched at the time points indicated in the figures with the addition of an equal volume of 2× SDS-loading buffer (LI-COR Biosciences) supplemented with 100 mM fresh β-mercaptoethanol. Samples were loaded onto 12% SDS-PAGE gels, transferred onto PVDF membranes (Bio-Rad), and immunoblotted with anti-Arp3 (1:5,000 dilution, Santa Cruz Biotechnology) or anti-Arp2 (1:200 dilution, Cell Signaling Technologies) antibodies. Membranes were imaged using a G-BOX scanner (Syngene) and densitometric analysis was performed using Image Lab (Bio-Rad). Mean and SD values were calculated from three or more independent experiments (SI Appendix, Fig. S4).

van Eeuwen T., Boczkowska M., Rebowski G., Carman P.J., Fregoso F.E, & Dominguez R. (2023). Transition State of Arp2/3 Complex Activation by Actin-Bound Dimeric Nucleation-Promoting Factor. Proceedings of the National Academy of Sciences of the United States of America, 120(33), e2306165120.

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Phagocytosis Assay with pHrodo Conjugates

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pHrodo E. coli (K12 strain) bioparticle conjugates (catalog no: P35366 for green florescence, and P35361 for red florescence) and an opsonizing reagent (catalog no: E2870) were purchased from Thermo Fisher Scientific (Carlsbad, CA). Cell culture media were purchased from Thermo Fisher Scientific. The antibodies for Western blotting included anti-ARP2 (catalog no: 5614), anti-p-cofilin (catalog no: 3313), anti-total cofilin (catalog no: 5175), anti-βPIX (catalog no: 4515), anti-mouse p-STAT3 (Tyr705, catalog no: 9131), and anti-total STAT3 (catalog no: 9139); these were obtained from Cell Signaling Technologies (Danvers, MA). Anti-β-actin antibodies (clone AC-15, catalog no: A5441), an anti-Rac1 magnetic bead conjugate (catalog no: 16-319), and anti-Rac1 antibodies (catalog no: 05-389) were purchased from Millipore-Sigma (St. Louis, MO). A Rac1 activation magnetic bead pulldown assay kit (catalog no: 17-10394) was purchased from Millipore-Sigma (St. Louis, MO). Infrared dye-labeled secondary antibodies were obtained from Li-Cor Biosciences (Lincoln, NE). The STAT3 inhibitor stattic (catalog no: sc-202818) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Rac1 inhibitor NSC 23766 (catalog no: 13196) was purchased from Cayman Chemicals (Ann Arbor, MI). A MemBrite cell surface staining kit was purchased from Biotium (catalog no: 30093-T, Fremont, CA).

Zhou M., Aziz M., Yen H.T., Ma G., Murao A, & Wang P. (2022). Extracellular CIRP dysregulates macrophage bacterial phagocytosis in sepsis. Cellular and Molecular Immunology, 20(1), 80-93.

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Western Blot Analysis of Actin Regulators

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Whole cell protein extracts were obtained by direct lysis in Lammelli's Buffer and boiled at 100°C for 10 min. Proteins were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (Amersham Biosciences, UK). Non-specific binding sites were blocked for 1 hour at room temperature with 5% non-fat dry milk in TBS-T. Membranes were incubated overnight at 4°C with anti-Rac1/Cdc42 (#4651), anti-Phospho-Rac1/Cdc42 (Ser71) (#2461), anti-ARP2 (#3128), anti-PAK1/2/3 (#2604), anti-Phospho-PAK1 (Thr423)/PAK2 (Thr402) (#2601), and anti-GAPDH (#2118) (Cell Signalling, Leiden, The Netherlands). All primary antibodies were used at 1:1000, washed 3 times with TBS-T, followed by incubation with HRP-conjugated anti-rabbit secondary antibodies (#7074, 1:1000, Cell Signalling, Leiden, The Netherlands). Protein complexes were visualized with a chemiluminescent detection kit (Novex ECL) and bands were quantified using ImageJ software.

Zeng N., Salker M.S., Zhang S., Singh Y., Shi B., Stournaras C, & Lang F. (2016). 1α,25(OH)2D3 Induces Actin Depolymerization in Endometrial Carcinoma Cells by Targeting RAC1 and PAK1. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 40(6).

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