Anti arf6

The following primary antibodies were used: anti-ARF1 (mouse [mo]; Santa Cruz Biotechnology [SCBT]); anti-ARF3 (mo; SCBT); anti-actin (rabbit [rb]; Sigma); anti-acetylated alpha-tubulin (mo; Sigma); anti-alpha-tubulin (mo; Sigma); anti-HSP70 (chicken [ck]; StressMarq); anti-detyrosinated alpha-tubulin (rb; Abcam, Inc.); anti-FLAG (mo; Thermo Fisher); anti-FLAG (rb; Sigma); anti-HA (chicken; Thermo Fisher); anti-pan-ARF (mo; Millipore); anti-ARF6 (rb; Cell Signaling); anti-ARF5 (mo; Abnova); anti-ARF4 (rb; ProteinTech); anti-giantin (rb; BioLegend); anti-GM130 (mo; Becton Dickinson [BD]); anti-lipopolysaccharide (anti-LPS; mo; Virostat); anti-CT813 (rb; T. Hackstadt); anti-IncA (rb; T. Hackstadt). Goat anti-rabbit and anti-mouse IgG–Alexa Fluor488, -555, and or -647–conjugated secondary antibodies, goat anti-chicken IgY Alexa Fluor555-conjugated secondary antibody, and donkey anti-rabbit and anti-mouse IgG–horseradish peroxidase (HRP)–conjugated secondary antibodies were purchased from Invitrogen. Donkey anti-chicken IgY–HRP–conjugated secondary antibody was purchased from Pierce.

ARF6-GTP pulldown assays were performed according to manufacturer’s instructions using the Arf6 Activation Assay Kit (Cell Biolabs). Briefly, Mel92.1 or Mel202 cells were grown in RPMI-1640/10% FBS to 80% confluency in 10 cm tissue culture dishes and then treated with 1 μM to 5 μM Tris DBA palladium or vehicle (0.1% DMSO in medium) for 3 h at 37° C/5% CO₂. Cells were lysed at 4° C in 1X lysis buffer (Cell Biolabs kit) supplemented with protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). Total cell lysate (0.5 mg) was incubated with GGA3-conjugated agarose beads for 1 hour, and then the beads were washed three times with 1X lysis buffer supplemented with protease and phosphatase inhibitors. Precipitates with beads (ARF6-GTP) and total cell lysates (total ARF6) were analyzed by western blot using 5% nonfat dry milk in PBST as a blocking agent, anti-ARF6 as a primary antibody (diluted 1/1000; Cell Signaling Technology), and a secondary antibody conjugated to horseradish peroxidase (diluted 1/5000; Jackson ImmunoResearch). Signals were detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore). Quantification was performed using scanning densitometry and ImageJ (NIH), and ARF6-GTP levels were normalized to total ARF6 levels.

The samples were lysed in RIPA buffer (P0013B, Beyotime, China) with protease and phosphatase inhibitor cocktails added (TargetMol, USA). Through SDS-PAGE, total protein was isolated and transferred to PVDF membranes (Merck Millipore, Darmstadt, Germany). Following an hour of blocking in 5% nonfat milk, the membranes were incubated overnight at 4℃ with the following primary antibodies: anti-CD63 (1:2000, Abcam), anti-TGS101 (1:1000, Abcam), anti-ARF6 (1:1000, Cell Signaling Tech), and antibodies against C1QC (1:1000, Biosis, Beijing, China) and C3B (1:1000, Biosis, Beijing). After washing, membranes were incubated for one hour at room temperature with HRP-conjugated secondary antibodies (1:2000, Cell Signaling Technology). Using an Immobilon Western Chemiluminescent HRP substrate, signals were detected (Millipore, Darmstadt, Germany).