Anti chmp2b

Transfected cells were fixed with 4 % paraformaldehyde (Noblechem; #PAR500) for 10 min. For immunostaining, the transfected cells were permeabilized with 0.1 % Triton X-100 (Merck & Co., Inc., Kenilworth, NY, USA; #108603) and then blocked with 3 % bovine serum albumin (Sigma-Aldrich Co. LLC, St. Louis, MO, USA; #A7906) in phosphate-buffered serum (Welgene, Inc., Daegu, Korea) for 1 h at room temperature. The primary antibodies [anti-FLAG M2 (Sigma-Aldrich Co. LLC, F1804), anti-CHMP2B (Abcam PLC, Cambridge, UK; ab33174), anti-TMEM106B (Proteintech Group, Inc., Chicago, IL, USA; #20995-1-AP), anti-cMyc (EMD Millipore, Billerica, MA, USA; #06-549) and secondary antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA; #715-545-150, #715-165-150, #711-165-152, and #711-485-152) were incubated for 1 h each at room temperature. All of the photomicrographs were captured with a confocal microscope (Carl Zeiss AG, Jena, Germany; LSM 510). Image analysis and colocalization analyses were performed using ImageJ program (NIH) .
GraphPad Prism 5 was used for statistical analysis. The values are presented as the mean ± SEM of three independent replicates. One-way ANOVA followed by Tukey’s multiple-comparisons test for statistical analysis of more than three data sets or Student-t-test for statistical analysis of two groups was selected.

Thapsigargin and BFA were purchased from Sigma-Aldrich (St Louis, MO, USA). Apoptozole was purchased from Millipore (Billerica, MA, USA). Anti-pendrin (G-19, epitope against C terminus), anti-HA, anti-Myc, anti-aldolase and anti-Flag antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-pendrin (ab66702, epitope against N terminus), anti-calnexin, anti-giantin, anti-Lamp1, anti-CHMP2B, anti-ATG7 and anti-Hsc70 were from Abcam (Cambridge, UK); anti-EEA1 was from BD Biosciences; anti-CD9 was from LSBio (Seattle, WA, USA); anti-DNAJC14 was from Novus (Littleton, CO, USA); anti-Rab7 was from Cell Signaling Technologies (Danvers, MA, USA); and anti-CFTR antibody (M3A7) was from Millipore. Detailed information for primary and secondary antibodies used in this study is summarized in Supplementary Table 4.

HIV-1 p24 was detected using anti-HIV-1 core antigen antibody-FITC (KC57-FITC; Beckman Coulter), and actin labeled with Cytopainter Phallodin-iFluor-555 (Abcam). Protein knockdown was detected by immunoblotting using rabbit anti-ARF1, anti-BIN1, anti-RAB8A, mouse anti-Rab7L1 (Abcam), and actin (Merck) antibodies, followed by secondary HRP-conjugated goat anti-rabbit and anti-mouse antibodies (Dako). Confocal microscopy was carried out using the primary antibodies anti-human CD81-APC (BD), anti-EEA1, anti-CHMP2B, anti-LAMP1, anti-Rab7, anti-Rab11, anti-Rab5 (Abcam). HRP uptake was detected using anti-HRP (Jackson Immunolaboratory). All unlabeled primary antibodies were detected with secondary anti-rabbit Alexa Fluor 546 (Life Technologies). The pharmacological inhibitors LY294002, bafilomycin A1, and indinivir (Sigma-Aldrich) were used at 50 μM, 0.5 μM, and 2 μg/ml, respectively.