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2 protocols using anti rab8

1

Protein Interaction Analysis Reagents

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Anti-FLAG and anti-Flag M1 agarose beads were from Sigma; anti-PPP2R1A and anti-Rab8, anti-Rab9 and Rab7 antibodies were from Cell Signaling. Anti-PPP2CA from Millipore, Inc. Peroxidase-conjugated anti-rabbit, anti-mouse and anti-goat secondary antibodies were from Jackson ImmunoResearch. Antibodies for immunofluorescence: anti-Rab8 and anti Rab9 were from Cell Signaling, anti-PPP2R1A was from Santa Cruz, anti-mouse Alexa Fluor-555-conjugated secondary antibody and anti-rabbit Alexa Fluor 488-conjugated secondary antibody were from Invitrogen. PPP2CA and PPP2R3C encoding plasmids were purchased from OpenBiosystem. PPP2R1A construct was kindly provided by Marc Vidal. RAB-GST cDNAs were kindly provided by Prof. Fukuda23 (link).
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2

Imaging Flow Cytometry for Immune Cell Analysis

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For imaging flow cytometry experiments: anti-CD8 APC-Cy7 (BD Biosciences, San Jose, CA or Tonbo Biosciences, San Diego, CA) and anti-CD56 PE-CF594 (BD Biosciences) were used for surface staining; all intracellular stains included anti-perforin D48 PE or BV421 (Biolegend, San Diego, CA) and anti-perforin δG9 FITC (Biolegend) and one of the following unconjugated antibodies: anti-CD71 (Cell Signaling Technology, Danvers, MA), anti-granzyme B (BD Biosciences), anti-rab3D (Abcam, Cambridge, MA), anti-rab4 (Pierce Antibodies, Waltham, MA), anti-rab5 (Cell Signaling Technology), anti-rab7 (Santa Cruz Biotechnology, Dallas, TX), anti-rab8 (Cell Signaling Technology), anti-rab11a (Cell Signaling Technology), anti-rab27a (Abcam), anti-rab35 (Abcam), anti-rab37 (Abcam), anti-syntaxin6 (Cell Signaling Technology), anti-syntaxin7 (R&D systems, Minneapolis, MN), anti-vti1b (Abcam), anti-VAMP3 (Abcam), anti-VAMP4 (Abcam), or anti-SNAP23 (Abcam). The unconjugated antibodies were detected using goat anti-rabbit AF647, chicken anti-goat AF647, or donkey anti-sheep AF647 secondary antibodies (Invitrogen, Carlsbad, CA). For confocal microscopy, perforin D48 FITC (Abcam), perforin δG9 AF647 (Biolegend), and goat anti-rabbit or donkey anti-sheep AF568 secondary antibodies (Invitrogen) were used, along with the selected rab or SNARE antibody.
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