Rabbit anti ha

For Western blot analysis cells were lysed in RIPA buffer and different amounts of total proteins were separated by electrophoresis through SDS-PAGE gels and blotted into nitrocellulose membranes. Mouse anti-RTN-1C (Abcam, ab8961) was diluted 1:1000 rabbit anti-LC3 (Cell Signaling, 3868 S) was diluted 1:1000, rabbit anti-syntaxin 17 (Abcam, ab116113) was diluted 1:200, rabbit anti-HA (Sigma, H6908) was diluted 1:2000. Rabbit anti-actin (Sigma, A2066) diluted 1:2000, mouse anti-GADPH diluted 1:1000 (Santa Cruz, sc-47724) and mouse anti-tubulin 1:2000 (Sigma, T-4026) were used as loading controls. Secondary antibodies (Biorad, 1721011, 1706515) were diluted 1:5000.

Cells (10⁴ cells per well) were grown on acid-washed eight-well glass slides (Thermo Scientific) in EMEM plus 10% FBS for 24 h. After treatment, paraformaldehyde-fixed cells were incubated with 4% BSA (1 h, 20°C) followed by overnight incubation with primary antibodies [rabbit anti-HA, 1:50, (Sigma); rabbit anti-TrkA, 1:50, (Alomone, Israel); mouse anti-CD44, 1:200, (Cell Signaling Technology)]. PLA was performed as recommended by manufacturer instructions.

HA-NIS-TPC1 cells were seeded in 100 mm dishes and allowed to grow to near 100% confluence. The cells were placed on ice, washed three times with ice-cold PBS⁺⁺, and lysed in 500 µL lysis buffer [50 mM Tris/HCl pH 7.5, 2 mM MgCl₂, 150 mM NaCl, 10% (v/v) glycerol, 1% (v/v) NP40, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA)]. The cell lysates were cleared at 10.000× g for 10 min at 4 °C. In order to reduce the non-specific binding, the lysates were pre-cleared by incubation with 40 μL G-protein agarose beads (Roche, Mannheim, Germany) for 60 min at 4 °C. The pre-cleared lysates were incubated overnight at 4 °C with 4 µg/mL of either rabbit anti-HA (Sigma-Aldrich, St. Louis, MO, USA) or goat anti-rabbit IgG (Bio-Rad; Hercules, CA, USA; control condition). Next, the lysates were rotated for 1 h at 4 °C with 30 µL Dynabeads G-Protein (Invitrogen, Carlsbad, CA, USA), and the immunoprecipitates were washed 5 times for 5 min with ice-cold wash buffer (50 mM Tris/HCl pH 7.5, 2 mM MgCl₂, 300 mM NaCl, 10% (v/v) glycerol, 1% (v/v) NP40). The proteins were recovered in 50 µL 2× modified Laemmli buffer and further analyzed by Western blotting or mass spectrometry.

Cells were fixed with 4% formaldehyde or ice-cold methanol and blocked with PBS/2% normal goat serum. Fixed cells were incubated with the primary antibodies rat anti-HA (Roche, 3F10; 1:300), rabbit anti-HA (Sigma-Aldrich, H6908, 1:300), mouse anti-Cortactin (MerckMillipore, 4F11; 1:500), mouse anti-E-cadherin (BD Biosciences, 610182, 1:1000) and rabbit anti-TKS5 (Santa Cruz, sc-30122, 1:100) as indicated in the text at 4°C overnight, followed by Alexa Fluor_ 488-conjugated donkey anti-rat IgG (1:500, A21208; Life Technologies), Alexa Fluor_ 350-conjugated goat anti-mouse IgG (1:500, A11045; Life Technologies), Alexa Fluor_ 594-conjugated goat anti-mouse IgG (1:500, A21135; Life Technologies) or Alexa Fluor_ 488-conjugated goat anti-rabbit IgG (1:500, A-11029; Life Technologies) for 1 h at room temperature and counterstained with DAPI (Molecular Probes).
F-actin was stained by Alexa fluor 488 phalloidin or Rhodamine phalloidin (1:100, A12379, R415, Life Technologies).

Cells were harvested and lysed with 500 μL of lysis buffer (150 nmol/L NaCl, 50 nmol/L Tris-HCl (pH 7.4), 1% Triton X-100, 1 mmol/L EDTA (pH 8.0), and 0.1% sodium dodecyl sulfate (SDS)) with a protease inhibitor cocktail and then incubated on ice for 30 min. The supernatants were collected by centrifugation at 4 °C at 12,000 ×g for 30 min and then boiled in 5× SDS-PAGE loading buffer for 10 min. The prepared samples were resolved on 10% SDS-PAGE and then transferred onto nitrocellulose membrane (GE Healthcare). The membrane was blocked with 5% skim milk powder in phosphate-buffered saline (PBS) with 0.1% Tween 20 (PBST) for 30 min before being incubated with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Thermo Fisher Scientific) for 1 h. The primary antibodies were used as follows: mouse anti-Myc (1:10,000, MBL), mouse anti-HA (1:10,000, MBL), mouse anti-Flag (1:10,000, Sigma), rabbit anti-HA (1:10,000, Sigma), rabbit anti-Flag (1:10,000, CST), rabbit anti-Myc (1:10,000, Sigma), rabbit anti-VP30 pSer29 antibody (1:1000, ABclonal), rabbit anti-Na/K ATPase (1:1000, ABclonal), and mouse anti-GAPDH (1:1000, ABclonal). The secondary antibodies, goat anti-rabbit IgG and goat anti-mouse IgG, were used at a 1:5,000 dilution.

Biotinylated eluates and the corresponding total extract fraction were electrophoresed in 10% polyacrylamide gels. Next, they were blotted onto a nitrocellulose membrane (0.4 μm, GE Health care) using a transfer buffer consisting of 30 mM Tris base, 190 mM glycine, and 20% methanol. After blocking at room temperature in TBS-T (20 mM Trizma base, 500 mM NaCl, 0.1% Tween-20) with 2% skimmed milk powder for at least 30 min, membranes were incubated overnight with rabbit anti-HA (Sigma, dilution 1:1000 in TBS-T 5% skimmed milk) or rabbit anti-Beta tubulin (Sigma, used as a loading control, dilution 1:5000 in TBS-T 5% skimmed milk) at 4 °C. After washing with TBS-T, membranes were incubated with the secondary antibody for 1 h at room temperature (Sigma, 1:10000, anti-rabbit HRP conjugated, diluted in TBS-T 5% skimmed milk). Finally, the immunoreactive bands were revealed using ECL Prime (GE Healthcare), visualized with the iBright1500 system and further analyzed with the iBright Analysis software (v3.0.1; Invitrogen). The results presented here come from four independent experiments.