Control short hairpin rna shrna

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.

USP21 and control short hairpin RNA (shRNA) were purchased from Sigma–Aldrich. The targeting sequence for shUSP21#1 was 5′-TCACTAAGGAAGAAGAGCT-3′. The targeting sequence for shUSP21#2 was 5′-AACCTAATGTGGAAACGTT-3′. Flag-USP21, Myc-FOXD1, truncated mutants of USP21 and FOXD1, HA-Ub, HA-Ub-K0, HA-Ub-K11, HA-Ub-K27, HA-Ub-K48, and HA-Ub-K63 overexpression plasmids were purchased from GeneChem. All transfections used Lipofectamine 3000 (Invitrogen, catalog L3000150) were performed following the manufacturer’s instructions. Cells transfected with Lipofectamine 3000 for stable cell lines were screened by puromycin.