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Importantly, ACAP2 (also known as centaurin ��2) is recruited to the plasma membrane in a Rab35-dependent manner and functions as a GTPase activating protein (GAP) for ADP-ribosylation factor 6 (ARF6) [25]. ARF6 belongs to the ARF family of small GTP-binding proteins [26, 27]. Like all small GTPases, ARF6 cycles between an inactive GDP-bound form and an active GTP-bound form. ARF6 regulates membrane trafficking between the plasma membrane and endosomal compartment [28, 29] and is involved in remodeling of the actin cytoskeleton [30]. It is noteworthy that ARF6 plays an important role in the internalization of several types of bacteria [31, 32]. In the case of Chlamydia caviae, ARF6 is activated during bacterial infection and controls bacterial invasion by actin remodeling [32]. Moreover, just like Rab35, ARF6 has been shown to regulate Fc��R-mediated phagocytosis [33]. Altogether, these findings make both Rab35 and ARF6 good candidates for regulating phagocytosis of zymosan, which is involved in various types of receptors. In the present study, we present evidence that Rab35/ACAP2/ARF6 are crucial components of zymosan internalization and the activation-inactivation cycles of Rab35 and ARF6 are required for this process. 2. Materials and Ku-0059436 molecular weight Methods 2.1. Reagents Bovine serum albumin (BSA), zymosan, and Dulbecco's modified Eagle's medium (DMEM) were obtained from Sigma Chemical (St. Louis, MO). Fetal bovine serum (FBS) was obtained from BioSolutions International (Melbourne, Australia). Monoclonal mouse anti-GFP antibody (GF200) (Nacalai Tesque) and monoclonal rabbit anti-ARF6 antibody (Cell Signaling Technology) were purchased from commercial sources. The other reagents were purchased from Wako Pure Chemicals (Osaka, Japan) or Nacalai Tesque (Kyoto, Japan), unless otherwise indicated. 2.2. Cell Culture Mouse macrophage RAW264 cells and COS-7 cells were cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100?U/mL penicillin and 100?��g/mL streptomycin, as described in the manuals of the cell line bank. RAW264 cells stably expressing Rab35 shRNA or GFP shRNA were maintained in the culture medium supplemented with 5?��g/mL puromycin, as previously described [21]. Before the imaging experiments, the culture medium was replaced with Ringer's buffer (RB) consisting of 155?mM NaCl, 5?mM KCl, 2?mM CaCl2, 1?mM MgCl2, 2?mM Na2HPO4, 10?mM glucose, 10?mM HEPES pH 7.2, and 0.5?mg/mL BSA. 2.3. DNA Constructs and Transfection pEGFP-Rab35, pTagRFP-Rab35, pEGFP-Rab35-Q67L, pTagRFP-Rab35-Q67L, pEGFP-Rab35-S22N, pEGFP-ACAP2, pEGFP- ACAP2-��ANKR, and pEGFP-ACAP2-RQ were described previously [21, 25]. pECFP-ACAP2 and pECFP-ACAP2-RQ were generated by the replacement of GFP with CFP. ARF6-wt-CFP, ARF6-Q67L-CFP, and ARF6-T27N-CFP were kindly donated by Dr. Joel A.