Anti lamp2 antibody

Cells were plated on coverslips and were transfected with shRNA for 48 h. Cells were then fixed for 15 min in 10% formalin and permeabilized for 5 min with 0.3% Triton X-100. After blocking in 10% normal goat serum, cells were stained using an anti-E-cadherin antibody (R&D Systems; 1:50) followed by an Alexa546-conjugated secondary antibody. EGF-treated transfected cells were fixed/permeabilized for 10 min in methanol. After blocking in 1% BSA, the cells were incubated with an anti-EEA1 (Cell Signaling; 1:100) or anti-LAMP2 antibody (Abcam, Cambridge, UK; 1:100) followed by a FITC-conjugated secondary antibody and then with an Alexa555-conjugated anti-EGFR antibody (Cell Signaling; 1:100) Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma) to visualize nuclei. Mounted samples were visualized with a confocal microscope (LSM 510 META; Carl Zeiss, Jena, Germany). Intensity of E-cadherin staining was quantitated by ImageJ software. Quantitative co-localization analysis of EGFR and EEA1 or LAMP2 was performed by calculating Pearson’s correlation coefficient (r) using ImageJ software.

Western blotting was conducted according to a protocol previously described [43 (link)]. Briefly, the tissues were lysed with radioimmunoprecipitation assay buffer (Applygen, Beijing, China) for 30 min and then homogenized using an ultrasound treatment at 100 W for 3 min and centrifuged at 12,000 × g for 20 min. Protein concentrations were quantified using the bicinchoninic acid assay. The proteins were then denatured with sodium dodecyl sulfate (SDS) protein loading buffer (Applygen) and were separated by SDS-polyacrylamide gel electrophoresis (15 μg per well). Samples were transferred to membranes and immunoblotted with the following primary antibodies: anti-GAPDH antibody (1:2000, Abcam, Cambridge, UK), anti-GAP43 antibody (1:1000, Abcam), anti-LAMP-2 antibody (1:500, Abcam), anti-SNAP25 antibody (1:1000, Abcam), anti-Alix antibody (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-TSG101 antibody (1:2000, ThermoFisher Scientific), anti-CD63 antibody (1:1000, ThermoFisher Scientific), and anti-APOB antibody (1:1000, ThermoFisher Scientific).

HeLa cells were seeded in 10cm dishes and transfected with siRNA against Luciferase or E6 for 72 hours. Optiprep gradients (5%-25%) were prepared as described previously [44 ] and equilibrated at room temperature for 3 hours. Cell extracts were prepared in the homogenization buffer containing protease inhibitors as described previously [44 ], and the lysates were syringed to ensure breakdown of the cells. The lysates were centrifuged at 3000 x g for 5 minutes at 4°C and the post nuclear extracts (supernatants) were loaded onto the equilibrated gradients. The gradients were centrifuged at 32,000 rpm for 18 hours at 4°C and fractions were collected using a peristaltic pump. The collected fractions were mixed with chilled acetone and incubated at -20°C overnight to precipitate the proteins. The precipitates were centrifuged at 14,000 rpm for 10 minutes at 4°C and washed once with chilled absolute ethanol. The precipitates were air dried and dissolved in 2x Laemmeli’s buffer. The fractions were loaded onto 12% SDS polyacrylamide gels and the endocytic profiles were analyzed by Western Blotting using anti-GLUT-1 antibody (Abcam), anti SNX27 antibody (Abcam), anti-p53 antibody (Santa Cruz), anti- Vps35 antibody (Abcam), anti- Rab4 antibody (Abcam) and anti- LAMP2 antibody (Abcam) as indicated.