Anti becn1

The human colorectal cancer cell lines HCT116 and SW480 were obtained from the ATCC (LGC Standards SLU, Barcelona, Spain). They were cultured in McCoy’s 5 A or Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Rockville, MD, USA) containing 10% fetal bovine serum (FBS), 100 units/mL penicillin, 100 µg/mL streptomycin (Invitrogen), and 2 mmol/L L-glutamine in a humidified atmosphere of 5% carbon dioxide in air at 37 °C. The fetal bovine serum was obtained from Corille (184590, Australia). 5-FU was obtained from Jinyao Amino Acid Co., Ltd. (Tianjin, China).
BafA1 (B1793), 3-methyladenine (M9281), chloroquine (C6628), the pancaspase inhibitor Z-VAD-FMK (C2105), necrostatin-1 (N9037) and SP600125 (S5567) were purchased from Sigma Aldrich. Anti-LC-3B (#3868), anti-p62 (#8025), anti-cleaved caspase-3 (#9661), anti-SQSTM1/p62 (#5114), anti-BECN1 (#3495), anti-SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (81E11) (#4668), anti-phospho-c-Jun (Ser63) (54B3) (#2361), and anti-GAPDH (#5174) antibodies were purchased from Cell Signaling Technology (CST). Anti-PCDH17 (HPA026817) for IHC was obtained from Sigma Aldrich. Anti-PCDH17 (ab128815) for WB was obtained from Abcam. The PCDH17 plasmid (NM_001040429) was purchased from OriGene.

The reagent rapamycin (Rapa; R0395; Sigma-Aldrich) was used in the study. The primary antibodies used were anti-ATG14 (no. 96752; Cell Signaling Technology), anti-UVRAG (no. 13115; Cell Signaling Technology), anti-BECN1 (no. 54101; Cell Signaling Technology), anti-p62 (P0067; Sigma-Aldrich), anti-LC3 (L8918; Sigma-Aldrich), anti-Bif1 (NBP2-24733; Novus), anti-GAPDH (RM2002; Beijing Ray Antibody Biotech), anti-RABV (5B12) (NB110-7542; Novus), and fluorescein isothiocyanate (FITC)-conjugated anti-RABV (800-092; FUJIREBIO). The secondary antibodies used were horseradish peroxidase (HRP)-conjugated goat antirabbit IgG (ab97051; Abcam) and goat antimouse IgG (sc-2005; Santa Cruz).

Cells were seeded in 60 mm tissue culture dishes and incubated for 24 h. To study the dose–response effect of lidocaine on cell cycle check-point protein expression and autophagy, the cells were treated with lidocaine (0.1, 0.25, 0.5, or 1 mM) with or without an inhibitor at 10 µM for 24 h. After washing the cells twice with ice-cold PBS, immunoblotting and total protein extraction were performed as previously described [21 (link)]. The primary antibodies included anti-phospho-GSK3β, anti-GSK3β, anti-ATG-5 and -7, anti-BECN1, anti-LC3B, anti-p27, anti-CCND1 (Cell Signaling Technology), and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Invitrogen, Carlsbad, CA, USA).

Seven weeks after transplantation, half of the brain tissues were homogenized in RIPA buffer (Thermo Fisher Scientific) containing protease inhibitors (GenDEPOT Inc., Barker, TX, USA) and sonicated. The homogenates were centrifuged at 20,000g for 20 min at 4 °C, and the supernatant was retained. For Western blot analysis of NEP, BECN1, LC3, and RAB7, protein samples were loaded onto NuPAGE 4–12% (w/v) and 12% (w/v) Bis-Tris Protein Gels (Thermo Fisher Scientific) and transferred to a polyvinylidene difluoride (PVDF) membrane (Roche, Mannheim, Germany). The membrane was blocked with 5% (w/v) milk and incubated with primary anti-NEP (1:1000, Merck Millipore, AB5458), anti-BECN-1 (1:1000, Cell Signaling Technology, Danvers, MA, USA, 3738S), anti-LC3 (1:1000, Cell Signaling Technology, 2775S), anti-RAB7 (1:1000, Proteintech Group Inc., Rosemont, IL, USA,55469-1-AP), and anti-β-actin (1:1000, Santa Cruz Biotechnology, SC47778) antibodies. The membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibodies and developed using enhanced chemiluminescence detection reagents (Thermo Fisher Scientific).

For western blotting analysis, total proteins were collected from striatum, SN, and BV2 cells and stored at −80 °C. 40 ug of total protein lysate was loaded onto a 12% sodium-dodecyl-sulfate polyacrylamide gel in each lane, then transferred onto a polyvinylidene-difluoride membrane (Millipore, United Ststes). The primary antibodies were incubated overnight at 4 °C included anti-TH (1:500; Santa Cruz, sc-25269), anti-solute carrier family 6 member 3 (SLC6A3) (1:1,000; ABclonal, A152360), anti-dopamine receptor D2 (DRD2) (1:1,000; ABclonal, A12930), anti-AIF1 (1:1,000; Santa Cruz, sc-32725), anti-GFAP (1:1,000; ABclonal, A14673), anti-NLRP3 (1:200; AdipoGen, AG-20B-0014-C100), anti-PYCARD (1:1,000; Immunoway, T0365), anti-CASP1 (1:1,000; ABclonal, A0964), anti-IL1B (1:1,000; ABclonal, A12688), anti-MAP1LC3B (1:1,000; ABclonal, A11282), anti-BECN1 (1:1,000; Cell Signaling Technology, 3738S) and anti-ACTB (1:3,000; ABclonal, AC026). HRP-conjugated anti-Rabbit antibody (1:5,000; ABclonal, AS014) or anti-Mouse antibody (1:5,000; ABclonal, AS003) was used as secondary antibody. ImageJ software was used to quantify the target bands and ACTB as an internal control.

Antibodies for β-Actin (#4970), p-mTOR (Ser-2448, #5536), p-ERK1/2 (Thr-202/Tyr-204, #4370), PARP (#9542S), caspase-3 (#9665S), anti-ULK1 (#8054S), anti-pRSK (#9344), anti-pATK (#9275S), anti-BECN1 (#3495S), cleaved caspase-3 (#9664S) and BAX (#2772S), ERK1/2 (#9102) and HRP-conjugated secondary anti-rabbit and anti-mouse antibodies (#7076 and #7074) were from Cell Signaling Technology (Beverly, MA, USA). Anti-P53 (#SC-6243), anti-UHRF1 (#SC-373750) and anti-P21 (#SC-397) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-human LC3B (#NB100-2220) and anti-P62 (#NBP1-42821) antibodies were obtained from Novus Biologicals (Littleton, CO, USA). Anti-ATPA3 (#ab2826) antibody was from Abcam (Cambridge, UK).

Cells were washed in PBS and then lysed in Triton X‐100 lysis buffer (20 mm Tris/HCl pH 7.5, 150 mm NaCl, 1% Triton X‐100, protease inhibitor EDTA free). After centrifugation, the protein content of the supernatants was measured using DC Protein Assay (Bio‐Rad Laboratories). Equal amounts of whole‐cell lysates were separated by SDS/PAGE and then electrotransferred onto polyvinylidene difluoride membranes. Membranes were probed with the following primary antibodies: anti‐VDUP‐1 (Invitrogen, 40‐3700, Carisbad, CA, USA), anti‐GAPDH (Santa Cruz Biotechnology, Inc., 0411, Dallas, TX, USA), anti‐LC3B (Cell Signaling Technology, #3738, Danvers, Essex, MA, USA), and anti‐BECN1 (Cell Signaling Technology, #2775). For secondary antibodies, HRP‐conjugated anti‐rabbit IgG and anti‐mouse IgG (GE Healthcare Life Sciences, Marlborough, MA, USA) were used. Blots were visualized using Chemi‐Lumi One Super (Nacalai Tesque) and ChemiDoc XRS + Imager (Bio‐Rad Laboratories, Hercules, CA, USA) according to the manufacturer's recommendations.