Image quant las 300

Oligonucleotide primers for convention PCR was synthesized by Integrated DNA technologies (Coralville, Iowa) and are listed in Table S1. Total RNA and proteins were isolated from various using standard protocols as we described^{11 (link)}. Total RNA was subjected to conventional PCR analyses using a master mix from Bio-Rad Laboratories (Hercules, CA). Protein extracts were subjected to immunoblot analysis using antibodies against PD-L1, EGFR, Alix, p-ERK, ERK, p-JNK, JNK, p-p38, p38, cyclin D1 (Cell Signaling Technology, Danvers, MA, USA), CD63, Her2 and GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase (Abcam, San Francisco, CA, USA). Immune complexes were detected with appropriate secondary antibodies from Jackson ImminoResearch Inc. (West Grove, PA, USA) and chemiluminescence reagents (Bio-Rad, Hercules, CA, USA) as described^{13 (link),44 (link)}. Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/).

Our standard laboratory protocol, described previously, was followed to prepare the cell lysates and to perform the immunoblots [41 (link)]. In brief, cells were lysed in phosphatase and protease inhibitors containing ice-cold NP40 lysis buffer (Invitrogen, Frederick, MD, USA). The supernatant was used for Pierce BCA protein assay (Thermo Scientific) and the proteins were separated using a 4–20% SDS-PAGE gradient gel and transferred onto a PVDF membrane. To block the non-specific binding sites, casein blocking buffer (Li-Cor, Lincoln, NE, USA) was used. The same buffer used to dilute primary antibodies. The membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (Sigma-Aldrich). After an overnight incubation with primary antibodies at 4 °C, membranes were washed and incubated again with respective secondary antibodies, either goat anti-rabbit IgG at 1:2500 dilution (#7074S, Cell Signaling Technology) or goat anti-mouse IgG at 1:5000 dilution (#7076P2, Cell Signaling Technology) at room temperature for 1 h. Chemiluminescence (LumiGLO, Gaithersburg, MD, USA) and autoradiography were used to visualize the final reaction. In some cases, immunoblot signals were captured using the ImageQuant Las 300 (GE Healthcare, Piscataway, NJ, USA) system. Densitometry of the immune-bands was performed using the ImageJ software (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/).

Protein extracts were subjected to immunoblot analysis using antibodies against Alix, p-ERK, ERK (Cell Signaling Technology, Danvers, MA, USA), GAPDH (Santa Cruz Biotechnology, Dallas, TX), Rab27a (Proteintech, Chicago, IL, USA) and nSMase2 (Santa Cruz Biotechnology). Immune complexes were detected with appropriate secondary antibodies from Santa Cruz Biotechnology and chemiluminescence reagents (Pierce, Rockford, IL, USA) as we described^{54 (link)}. Immunoblot signals were captured using the Image Quant Las 300 (GE Healthcare, Piscataway, NJ, USA). Densitometric analysis was performed using ImageJ (NIH, Bethesda, MD, USA, http://imagej.nih.gov/ij/).