Anti myc tag

Anti-ROS1 (#3266, 1:1000 for immunoblotting, 1:100 for immunofluorescence staining) and anti-DRP1 (#8570, 1:1000 for immunoblotting) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-GAPDH (#G8795, 1:5000 for immunoblotting) and anti-myc tag (#05-724MG, 1:1000 for immunoblotting) antibodies were purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-TOM20 (#sc-11415, 1:1000 for immunoblotting, 1:100 for immunofluorescence staining) and anti-TIM23 (#sc-514463, 1:1000 for immunoblotting) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Secondary antibodies conjugated to Alexa Fluor 700 (#A21036), Alexa Fluor 488 (#A11001), Alexa Fluor 555 (#A21428), or Alexa Fluor 647 (#A21235) were purchased from Invitrogen (Carlsbad, CA, USA) and diluted 1:10,000. Rhodamine phalloidin (#A12379) was purchased from Invitrogen and diluted 1:1000 for immunofluorescence staining. IRDye800CW-labeled anti-rabbit secondary antibody (#926-32211, 1:10,000 for immunoblotting) was purchased from LI-COR Biosciences (Lincoln, NE, USA). Anti-GFP tag (#GTX113617, 1:1000 for immunoblotting) and anti-mCherry (#GTX59788, 1:1000 for immunoblotting) antibodies were purchased from GeneTex (Irvine, CA, USA). Foretinib (#A2974) was purchased from ApexBio Technology (Houston, TX, USA).

Immunofluorescence analysis was performed in H1299 cells fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with Triton X100 0.2% (Sigma-Aldrich) and blocked with 3% BSA (all from Sigma-Aldrich). Cells were stained with anti-Myc-Tag (9E10, Sigma-Aldrich) - for INSL4 analysis - and anti-calreticulin (StressGen Biotechnologies Corp., BC, Canada) antibodies. Goat anti-Mouse IgG (H+L) Alexa Fluor^® 555 conjugate and Goat anti-Rabbit IgG (H+L) Alexa Fluor^® 488 conjugate secondary antibodies were used respectively (Thermo Fisher Scientific, Waltham, MA, USA). DNA was stained with DAPI (Sigma-Aldrich). Cells alternatively stained with a fluorescent phalloidin-FITC conjugate (Sigma-Aldrich) solution in PBS at room temperature for 40 minutes. Phalloidin-FITC conjugate were used as a cytoskeleton marker.
Fluorescence analysis was performed using Zeiss Axioplan (Zeiss, Oberkochen, Germany) fluorescence microscope controlled by Spot-2 cooled camera (Diagnostic Instruments, Sterling Heights, USA). Experiments were performed at least three times and performed each time in triplicate. Images were exported in TIFF, and contrast and brightness were adjusted in Corel Paint Shop Pro X9 and final figures were generated with Adobe Illustrator 6.

Cellular or tissue samples were lysed with RIPA lysis buffer freshly adding cocktail protease inhibitor (Thermo Scientific). Equal amounts of cellular proteins were subjected to electrophoresis in SDS-PAGE, and transferred to nitrocellulose membranes (Millipore). The membranes were blocked and then incubated at 4 °C overnight with indicated first antibodies, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Thermo Scientific). The protein bands were detected and analyzed with an ECL. The following antibodies were used: anti-Aurora A (Upstate, #07-648), anti-phospho-Aurora A (Thr288) (Cell Signaling Technology, #3079), anti-YAP (Cell Signaling Technology, #4912), anti-phospho-YAP(ser127) (Cell Signaling Technology, #4911), anti-phospho-YAP(Ser397) (Cell Signaling Technology, #13619), anti-Lats1 (Cell Signaling Technology, #3477 P), anti-phospho-Lats1 (Thr1079) (Cell Signaling Technology, #9654P), anti-Lats2 (Rui Ying biological, China, #RLP1047), anti-SAV1 (Cell Signaling Technology, #3507P), anti-MST1 (Cell Signaling Technology, #3682P), anti-GAPDH (Kangcheng, China,#KC-5G4), anti-Lamin B1 (Epitomics, #6581-1), anti-CTGF (Life Science Products & Services, #AB60212a), anti-Myc Tag (MERCK, 05-724).

Cells were lysed on ice for 30 min with RIPA buffer, protease inhibitor cocktail ± phosphatase inhibitor cocktail. Cell lysate proteins (80 μg) were resolved on SDS-PAGE and transferred to nitrocellulose for western blots. Blots were quantitated by densitometry using ImageJ (NIH, Bethesda, MD, United States) and normalized to cellular Actin. Antibodies against LAMP-2A (Cat #ab18528), LAMP-2B (Cat #ab18529), HSP90 (Cat #ab13494), and cathepsin A (Cat #ab79590) were from Abcam (Cambridge, MA, United States). Chk1 (Cat #2360), phospho-Chk1 (Ser345) (Cat #2341), IκBα (Cat #4814), phospho-IκBα (Ser32/36) (Cat #9246), LC3B (Cat #2775), and histone H3 (Cat #3638) were from Cell Signaling Technology (Danvers, MA, United States). LAMP-2 (Cat #H4B4-c) was from DSHB (Iowa City, IA, United States) and HSC70 (Cat #ADI-SPA-815) from Enzo Life Sciences (Farmingdale, NY, United States). Anti-Myc Tag (Cat #05-724) and cathepsin D (Cat # IM03) were from EMD Millipore (Billerica, MA, United States). Cathepsin B (Cat # sc-13985), p53 (Cat # sc-126), and p21 (Cat # sc-756) were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Actin (Cat # MS-1295-P0) was from Thermo Fisher Scientific.

Before gel electrophoresis the cell lysate was supplemented with Bolt reducing agent and Bolt gel loading buffer (both Thermo Fisher Scientific, MA, USA) and incubated at 90 °C for 5 minutes. The samples were then separated by SDS-PAGE (Bolt Gels, Thermo Fisher Scientific, MA, USA), followed by transfer onto nitrocellulose membrane, which were blocked in 3% skimmed milk in PBS + 0.1% Tween (PBS-T) for 1 hour at room temperature. Immunoblotting was performed at 4 °C overnight using the following primary antibodies diluted in blocking buffer: phospho/total p44/42 MAPK, phospho/total Akt, phospho/total PLCgamma1 (all at 1:1000 and all from Cell Signaling Technology, USA), or anti-Myc-tag (1:1000; Merck Millipore, MA, USA). The following day the membrane was washed three times in PBS-T and then incubated with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG or anti-mouse IgG at 1:5000, from Agilent Technologies, CA, USA) diluted in blocking buffer for 4 hours at 4 °C. This step was followed by another 3 washes in PBS-T and finally chemiluminescent development using the ECL Western Blotting Substrate (Promega, WS, USA). The signal was captured with the BioRad ChemiDoc Imager (Bio-Rad Laboratories, CA, USA) and bands quantified using ImageJ (https://imagej.nih.gov/ij/).

Whole cell extracts were prepared by rotating the cell pellets for 2 hrs at + 4°C in five volumes of the high salt lysis buffer (50 mM Tris, 300 mM NaCl, 10% glycerol, 0.5% NP-40, 1x complete ULTRA protease inhibitors (Roche)). Proteins were resolved on 10% SDS-PAGE gels, transferred to the Immobilon-P/E PVDF membrane (Merck Millipore), and immunodetected using the SuperSignal West Pico PLUS Chemiluminescent Substrate (ThermoFisher Scientific) and the following antibodies: anti-FLAG M2 (Sigma, F1804, 1:1000), anti-DYKDDDDK Tag (Cell Signaling, 14793, 1:1000), anti-Myc Tag (Millipore, 05–724, 1:1000), anti-mouse-HRP (GE healthcare, NA931-1ML, 1:5000), anti-rabbit-HRP (GE healthcare, NA934-1ML, 1:5000).