Rab11

Normal human adult kidney samples were collected from kidneys nephrectomised due to renal cancer (Karolinska University Hospital, Stockholm, Sweden). Nephrin and podocin antibodies have been described previously^{9 (link),23 (link)}. Other antibodies were: Med22—Atlas Antibodies and Sigma; Vimentin, alpha SMA, WT-1—Sigma (human); CD31—Abcam; Clathrin, Caveolin, Lc3II, Atg16L, Atg5, Beclin, Rab5, Rab7, Rab11 (Cell Signalling); Synaptopodin, Rab3b and LAMP2 (Santa Cruz), mouse nephrin (Acris), mouse WT1 (Millipore).
The samples were snap-frozen, and the cryosections (10 μm) were post-fixed with cold acetone (− 20 °C) followed by blocking in 5% normal goat serum. The primary antibodies were incubated overnight at 4 °C, followed by a 1-h incubation with the secondary antibody. For double-labelling experiments, the incubations were performed sequentially.

Cells were lysed in RIPA buffer (Tris pH 7.4 50mM, NaCl 150mM, NP-40 1%, SDS 0.1%, EDTA 2μM) containing proteinase inhibitors (Roche) and phosphatase inhibitors (Roche). The cell lysates (20μg protein) were subjected to SDS-PAGE and transferred to polyvinylidenedifluoride membranes. The membranes were blocked with 5% non-fat milk in TBST and incubated with specific primary antibodies. Detection was performed using SuperSignal WestPico chemiluminescent substrate (Thermo Scientific) followed by exposure to X-ray film. Antibodies against the following proteins were used: pAKT, pERK, pS6, Rab11, EGR1, cleaved PARP, cleaved Caspase3, BIM, pBAD and actin (Cell Signaling Technology).

Cells were lysed in RIPA lysis buffer (0.5M Tris-HCl, pH 7.4, 1.5M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA, protease inhibitor), and the protein lysates were subjected to SDS-PAGE followed by electroblotting onto a PVDF membrane. Membranes were probed with the following primary antibodies: α-galactosidase A, TSG101 (GeneTex, Irvine, CA, USA), CD63 (Santa Cruz Biotechnology, Dallas, TX, USA), Calnexin (Abcam, Cambridge, MA, USA) GDRID2, VPS36, VTI1A (Proteintech Group, Wuhan, Hubei, P.R.C), LC3 (Novus Biologicals, Centennial, CO, USA), Rab11 and GAPDH (Cell Signaling Technology, Denver, MA, USA). The bands were visualized by chemiluminescence detection reagents.

U251n cells on coverslips were washed twice with phosphate buffered saline (PBS). The cells were then fixed for 20 minutes at room temperature with solution containing 4% paraformaldehyde and 4% sucrose in PBS, following previously published protocol.10 Three washes with PBS were used to remove the fixing solution. Cells were then incubated for a half‐hour in block solution (1% BSA, 0.3 mol/L glycine, and 0.1% Tween 20). For co‐localization experiments with NHE9‐GFP, primary antibodies Rab 5 (Cell Signaling Technology), Rab 11 (Cell Signaling Technology) and LBPA (Echelon) were diluted 1:100 in block solution without Tween 20 and incubated overnight at 4°C. Following PBS washes, Alexa Fluor‐conjugated secondary antibodies (Invitrogen) were used at 1:1000 dilutions for 30 minutes. Cells were mounted onto slides using Prolong gold antifade reagent (Invitrogen). Immunostaining of human brain microvascular endothelial cells (BMVECs) in culture with RAW264.7 cells was conducted as described previously.10 Anti‐human von Willebrand factor antibody (DakoCytomation) was used as a marker for BMVECs. All slides were imaged using Lumascope‐620 microscope (Etaluma).

The antibodies used were as follows: α-adaptin (Becton Dickinson); GluA1 (Millipore); GluA2 (rabbit Synaptic Systems, mouse Becton Dickinson); GluA3 (Alomone); LAMP1 (Abcam); PICK1 (rabbit Abcam, mouse Neuromab); tubulin (Sigma); Rab5 (Cell Signaling); Rab11 (Cell Signaling).

For co-immunoprecipitation, 4mg of total protein lysate were subjected to GFP-mediated immunoprecipitation of YFP and fusion proteins using GFP-Trap® agarose beads (ChromoTek, Planegg-Martinsried, Germany). Superior expression of YFP compared to EpCAM-YFP was adjusted through the addition of wild-type mF9 cell lysate to YFP lysates. Precipitated proteins were washed with ice-cold washing buffer containing 0.5% tween ×100 in TBS, resuspended in 20μL Laemmli buffer (Laemmli, 1970 (link)), separated in a 10%-SDS-PAGE, transferred onto activated PVDF membrane (Millipore, Darmstadt, Germany) and detected with GFP (Santa Cruz, sc-9996; USA), prohibitin-1 (Santa Cruz, sc-28259; USA), prohibitin-2 (Santa Cruz, sc-67045; USA), calnexin (Enzo, ADI-SPA-860; USA), Rab5 (Cell Signaling, E6N8S; USA), Rab7 (Cell Signaling, D95F2; USA) and Rab11 (Cell Signaling, D4F5; USA) specific antibodies in conjunction with HRP-conjugated secondary antibodies and ECL (Millipore, Darmstadt, Germany).