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2 protocols using mouse anti eea1

1

Subcellular Localization and Trafficking Analysis

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Antibodies used for immunofluorescence microscopy or western blotting were obtained from commercial sources. The following antibodies were used: rabbit anti-NHE6 (Abcam; 137185), rabbit anti-NHE9 (Abcam; 167157), mouse anti-RACK1 (BD Biosciences; 610177), rabbit anti-RACK1 (Cell Signaling Technology; 5432), mouse anti-tubulin (Sigma-Aldrich; T6199), rabbit anti-actin (Sigma-Aldrich; A5060), mouse monoclonal anti-HA (BioLegend; MMS-101P), mouse anti-EEA1(Santa Cruz Biotechnology; 6415), mouse anti-Rab7 (Santa Cruz Biotechnology; 10767), rabbit phospho-(Ser) PKC substrate (Cell Signaling Technology; 2261), mouse monoclonal PKC (Santa Cruz Biotechnology; 80), mouse monoclonal anti-LAMP2 (Abcam; 25631), rabbit anti-p-glycoprotein (Abcam; 129450). All Alexa Fluor secondary antibodies were acquired from Thermo Fisher Scientific. 4′, 6-diamidino-2-phenylindol dilactate (DAPI), Alexa Fluor-conjugated Tfn and Lysotracker were purchased from Thermo Fisher Scientific. Chemotherapeutic drugs (Dox, Mtx, Dau) were obtained from the local drug dispensary of the Centre Hospitalier Universitaire de Sherbrooke. Cq and Baf were obtained from Sigma-Aldrich. Cq was dissolved in water and Baf in dimethylsulfoxide. Control short hairpin RNA (shRNA) and shRNA directed against human NHE6 or NHE9 were purchased from Sigma-Aldrich. RNA interference directed against RACK1 was purchased from Ambion.
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2

Immunofluorescence Staining of Organelles

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Mouse anti-HA HA.11 clone 16B12 (#MMS-101P, Covance, 1:1000). Rabbit anti-EGFR
(#HPA018530, Sigma, 1:100). Mouse anti-EEA1 (#SC-6415, Santa-Cruz, 1:200). Rabbit anti-LAMP2 (#H4B4, DSHB, 1:200). Rabbit anti-Rab7 (#9367, Cell Signalling, 1:100). Rabbit anti-HA (#3724, Cell Signalling, 1:1600). Rabbit anti-cathepsin D (#ab72915, Abcam, 1:100). Mouse anti-transferrin receptor (#13-6800, Life Technologies, 1:300). All secondary antibodies for IF were from Life Technologies and were used at a dilution of 1:400.
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