Anti ulk1

The following primary antibodies were used: anti-GAPDH (Sigma-Aldrich, G9545; 1:5000) anti-TOMM20 (Santa Cruz Biotechnology, SC-11415; 1:500), anti-FIS1 (Proteintech, 10956–1-1ap; 1:100), anti-ATP5F1A/ATP5A (Abcam, Ab14748; 1:200), anti-PDHA1 (Abcam, ab110330; 1:200), anti-ATG7 (Cell Signaling Technology, 8558; 1:1000), anti-RAB9A (Cell Signaling Technology, 5118; 1:1000), anti-ULK1 (Cell Signaling Technology, 8054; 1:1000), anti-RAB5A (Cell Signaling Technology, C8B1; 1:1000), anti-RAB7 (ERP7589; Abcam, ab137029; 1:1000), anti-LC3B (Sigma-Aldrich, L7543;1:200). Alexa Fluor 647‐conjugated goat anti-rabbit and anti-mouse IgG (Invitrogen, A21244 and A32728; 1:500) were used as secondary antibodies.

Before treatments, the cells were synchronized by serum-free media. The treated cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8 for 30 min. Protein content of the cell lysates was measured with Pierce BCA Protein Assay (Thermo Scientific, 23,225)^{39 (link),40 (link)}. The samples were separated on 10% or 15% sodium dodecyl sulfate polyacrylamide gel and the SDS-PAGE was done by using Hoefer miniVE (Hoefer Inc.) Proteins were transferred to 0.45 µM PVDF membrane (Thermo Scientific, 88,518). The membranes were blocked with TBS Tween (0.1%), or 5% non-fat dry milk or 1% bovine serum albumin (Sigma-Aldrich, A9647)^{39 (link),40 (link)}. The membranes were incubated with the following antibodies: antiLC3B (SantaCruz, sc-271625), antip62 (Cell Signaling, 5114S), antiAMPK-P (Cell Signaling, 2531S), antiAMPK (Cell Signaling, 2603S), antip70S6K-P (Cell Signaling, 9234S), antip70S6K (SantaCruz, sc-8418), antiULK1-555-P (Cell Signaling, 5869S), antiULK1-777-P (Merck, ABC123), antiULK1 (Cell Signaling, 8054S) and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (Cell Signaling, 7074S, 7076S). The bands were visualized using a chemiluminescence detection kit (Thermo Fisher Scientific Inc., 32,106)^{39 (link),40 (link)}.

Protein sample preparation and Western immunoblotting were performed as previously described (Qin et al., 2011 (link); Pandey et al., 2017 (link)). Primary antibodies used in the immunoblotting analysis included anti-p-ULK1, anti-ULK1, anti-ASK1, anti-p-ASK1, anti-SAPK/JNK, anti-p-SAPK/JNK (Cell signaling); anti-AMPKα, anti-p-AMPKα, anti-Atg9a (Thermo Scientific); anti-LC3, anti-p-ASK1 and anti-GAPDH (Santa Cruz Biotech., Inc); anti-BAK, anti-BAX (EMD Millipore), anti-p-IRE1α (GeneTex, Inc), anti-IRE1α (Novus Biologicals). Dilution of primary antibodies was 1:1,000. Secondary antibody HRP anti-IgG (Sigma-Aldrich, USA, 1:1,000~5,000) was used in the immunoblotting analysis. Densitometry of blots was performed using the ImageJ software package (http://rsbweb.nih.gov/ij/). The relative expression levels of target proteins and the ratio of blot LC3-II/LC3-I at the indicated time points were calculated as previously described (Qin et al., 2011 (link)). All Westerns were performed in triplicate and representative images are shown.

Cells were lysed with RIPA buffer (#P0013C, Beyotime, China). Protein concentration was quantified using a BCA protein assay kit (#P0012S, Beyotime, China) and transferred to PVDF membrane (# 88518, Thermo Fisher Scientific, USA) after separation on SDS-PAGE gels. The membranes were incubated with primary antibodies at 4°C for 12 h, and then washed three times with TBST buffer. Finally, the membranes were incubated with HRP-conjugated secondary antibodies for observation. An image processing system (Bio-Rad) and ImageJ software were used to determine and quantify these protein bands, respectively. Primary antibodies and a secondary antibody, including anti-GBP1 (#PA5– 23509), were purchased from Thermo Fisher Scientific. The other antibodies, including anti-GAPDH (#5174), anti-LC3A/B (#12741), anti-Beclin-1 (#3738), anti-AMPKa (#5831), anti-phospho-AMPKα (#50081), anti-mTOR (#2983), anti-phospho-mTOR (#2971), anti-ULK1(#8054), anti-phospho-ULK1 (#6887) and anti-rabbit IgG-HRP-linked antibody (#7074) were purchased from Cell Signaling Technology. The ratio of target protein/GAPDH or LC3II/LC3I is shown below the protein band.

Anti-ACTIN, Anti-ULK1, Anti-p-ULK1, Anti-p62, and Anti-LC3B were purchased from Cell Signaling Technology (Danvers, MA). The SEMA3C and GAPDH antibodies were purchased from Proteintech (Rosemont, IL). The Anti-KRAS antibody was from ABclonal (Woburn, MA). CellTiter-Glo^®2.0 was purchased from Sigma-Aldrich (St. Louis, MO). ECL Chemiluminescence kit was obtained from National Diagnostics (Atlanta, Georgia). Recombinant Human SEMA3C protein was purchased from the R&D Systems (Minneapolis, MN). EasySep™ Mouse CD8+ T Cell Isolation Kit was from Stem Cell (Cambridge, MA). Human KRAS (G12D) Expression Lentivirus was from GenTarget Inc (San Diego, CA). SEMA3C plasmids were purchased from Sino Biological (Wayne, PA). The SEMA3C inhibitor, 3,5,4’-Tribromosalicylanilide (CAS: 87-10-5), was purchased from TCI chemicals (Portland, OR) (26 (link)).

The following antibodies were obtained from Cell Signaling Technology (Beverly, MA): anti-pan-acetylated lysine (#9441), anti-MAP1LC3/LC3 (#4108), anti-RPS6KB/p70S6K (#9202), anti-phospho-RPS6KB/p70S6K (Thr389) (#9205), anti-TSC2 (#9442), anti-phospho-ULK1 (Ser 757) (#14202), anti-ULK1 (#8054), anti-phospho-DNM1L/Drp1 (Ser 616) (#3455), anti-phospho-MAPK3/ERK1-MAPK1/ERK2 (Thr 202/Tyr 204) (#9101), anti-MAPK3/ERK1-MAPK1/ERK2 (#9102), and anti-P-EIF2A/P-eIF2-α (Ser 51) (#9721). From Mitosciences: anti-RIESKE (MS305/C0183). From Abcam: anti-MFN2 (ab56889) and anti-HADHA (ab54477). From Sigma-Aldrich: anti-ACTB/β-actin (A5316). From Santa Cruz Biotechnology: anti-GFP (sc-9996), anti-TSC2 (sc-893), anti-TOMM20 (sc-17764), and anti-MFN1 (sc-50330). From Merck-Millipore: Nitrotyrosine 06-284. Other antibodies were used as follows: anti-mono- and polyubiquitinylated proteins FK2 conjugated with peroxidase (HRP) or FK2H from Enzo Life Sciences (BML-PW0150), anti-OPA1 (612606), and anti-DNM1L/Drp1 (611112) from BD Biosciences. Chloroquine C6628, propidium iodide (P4170), and thapsigargin (T9033) were from Sigma-Aldrich; rapamycin (553210) and resveratrol (R5010) were from Merck; and geneticin (G418) was from Santa Cruz (sc-29065).

Cells were harvested and lysed with 20 mM Tris, 135 mM NaCl, 10% glycerol, 1% NP40, pH 6.8. Protein content of cell lysates was measured using Pierce BCA Protein Assay (Thermo Scientific, Waltham, MA, USA, 23225). During each procedure equal amounts of protein were used. SDS-PAGE was done by using Hoefer miniVE (Amersham, UK). Proteins were transferred onto Millipore (Billerica, MA, USA) 0.45 µm PVDF membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and for antibody solutions. Loading was controlled by developing membranes for GAPDH or dyed with Ponceau S in each experiment. The following antibodies were applied: antiLC3B (SantaCruz, Santa Cruz, CA, USA, sc-16755), antiPARP (Cell Signaling, Danvers, MA, USA 9542S), antiGADD 153 (SantaCruz, sc-7351), antiCREB-2 (SantaCruz, sc-200), antiP-c-Jun (Cell Signaling, 9261S), antic-Jun (Cell Signaling, 9165S), antiPERK (Cell Signaling, 3192S), antiULK1 (Cell Signaling, 8054S), antiP-ULK1 (S555) (Cell Signaling, 5869S), antieIF2α (Cell Signaling, 9722S9), antiP-eIF2α (Cell Signaling, 9721L), and antiGAPDH (Santa Cruz, 6C5), HRP conjugated secondary antibodies (SantaCruz, sc-2020 and Cell Signaling, 7074S, 7076S).