Gapdh d16h11

The following antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA): Phospho‐KIT (Tyr703; D12E12) rabbit mAb (no. 3073), Phospho‐KIT (Tyr719) antibody (no. 3391), Phospho‐anti‐KIT (pY823) rabbit mAb (no. 77522), KIT (Ab81) mouse mAb (no. 3308), Phospho‐AKT (Thr308; 244F9) rabbit mAb (no. 4056), Phospho‐AKT (Ser473; D9E) XP rabbit mAb (no. 4060), AKT (pan; C67E7) rabbit mAb (no. 4691), Phosphop44/42 MAPK (ERK1/2; Thr202/Tyr204; 197G2) rabbit mAb (no. 4377), p44/42 MAPK(ERK1/2; 137F5) rabbit mAb (no. 4695), PhosphoSTAT3 (Tyr705; D3A7) XP rabbit mAb (Biotinylated; no. 4093), STAT3 (D3Z2G) rabbit mAb (no. 12640), GAPDH (D16H11) XP rabbit mAb (no. 5174), Phospho‐FRS2 (Tyr196) antibody (no. 3864), Phospho‐Histone H2A.X (Ser139; 20E3) rabbit mAb (no. 9718), PARP (46D11) rabbit mAb (no. 9532), and caspase‐3 (8G10) rabbit mAb (no. 9665). β‐actin antibody was purchased from TransGen Biotech (Beijing, China; no. HC201‐02).

Fenretinide, ABT-263, and Q-VD-OPh were purchased from ApexBio Tech (Houston, TX). Antibodies for BIM, BAX, BAK, BCL-X_L, Cleaved-PARP (Asp214), Cleaved-Caspase3, GAPDH (D16H11), HRP-linked Anti-Rabbit IgG, HRP-linked anti-mouse IgG, and Anti-Rabbit IgG conformation-specific antibodies were from Cell Signaling Technology (Beverly, CA); NOXA (114C307.1) was from Thermo Fisher Scientific (Waltham, MA); MCL-1 (ADI-AAP-240-F) was from Enzo Life Sciences (Farmingdale, NY); BCL-2 (100) was from Sigma-Aldrich. BAK antibodies for immunoprecipitation were purchased from Sigma-Aldrich (06–536 and AM03). ECL2 Western blotting substrate was purchased from Thermo Fisher Scientific (Rockland, IL). Annexin V-FITC and Annexin V-APC were purchased from Biolegend (San Diego, CA) and Thermo Fisher Scientific, respectively. Propidium Iodide was purchased from Sigma-Aldrich.

LM8 and 143B cells were collected and cell lysates were prepared using a CelLytic MT cell lysis reagent (Sigma-Aldrich, St. Louis, MI, USA) according to the manufacturer’s instructions. Western blotting was then performed to measure protein expression. Equal amounts of protein from each sample were analyzed as previously described [54 (link)], by immunoblotting with primary antibodies against cleaved caspase-3 (Asp175) (5A1E) (#9664, 1:1000), cleaved caspase-8 (Asp387) (D5B2) (#8592, 1:1000), phospho-P70-S6 (Thr389) (108D2) (#9234, 1:1000), P70-S6 (49D7) (#2708, 1:1000), phospho-RICTOR (Thr1135) (D30A3) (#3806, 1:1000), RICTOR (53A2) (#2114, 1:1000), LC3-I/II (D3U4C) (#12741, 1:1000), and GAPDH (D16H11) (#5174, 1:1000) obtained from Cell Signaling Technology (Danvers, MA, USA); and p62 (SQSTM1) (PM045, 1:1000) obtained from MBL International Corporate (MBLI) (Woburn, MA, USA), were used. Images were captured using a LAS-4000 camera system from Fujifilm (Tokyo, Japan) and quantified using ImageJ 1.52a (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

LM8 and 143B cells were collected, and cell lysates were prepared using a CelLytic MT cell lysis reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Western blotting analysis was performed as previously reported [17 (link)]. In short, equal amounts of protein from each sample were analyzed by immunoblotting with primary antibodies against cleaved caspase-3 (Asp175) (5A1E) (#9664, 1:1000), caspase-3 (#9662, 1:1000), LC3-A/B (D3U4C) (#12741, 1:1000), LC3-B (D11) (#3368, 1:1000), and GAPDH (D16H11) (#5174, 1:1000) (Cell Signaling Technology, Danvers, MA, USA). Images were captured using a LAS-4000 camera system from Fujifilm (Tokyo, Japan) and quantified using ImageJ 1.52a (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).

Homogenized tissues were lysed for protein extractions. Protein extracts (30–50 mg) were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes; incubated with rabbit anti-GPER (ab39742; from Abcam, Cambridge, Massachusetts) as a primary antibody; and then developed with a secondary anti-rabbit antibody (obtained from Cell Signaling Technology, Danvers, Massachusetts). The signal was detected using a LI-COR Biosciences Odyssey Infrared Imaging System (LI-COR, Lincoln, Nebraska). Equivalent protein loading and transfer efficiency were verified by staining for GAPDH (D16H11, from Cell Signaling Technology, Danvers, Massachusetts).

CRC cells (SW480 and SW620) were transfected with NNT-AS1 siRNAs or Control siRNA in 6-well plates. Seventy-two hours after transfection, the cells were washed twice with cold PBS, and 30 μL of RIPA (Solarbio, Shanghai, China) containing a protease inhibitor cocktail (Invitrogen, Carlsbad, CA, USA), a phosphatase inhibitor cocktail (Invitrogen, Carlsbad, CA, USA) and 2 mM ethylenediaminetetraacetic acid (EDTA) at pH 8.0 was added to each well. Following this, the cells were collected into a cold tube. Cell lysates were centrifuged at 12,000 g for 20 min at 4°C. Subsequently, the proteins in the lysates were separated on a 10% polyacrylamide gel and transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 10% non-fat milk for 4 h at room temperature and then incubated with primary antibodies for 1 h at 4°C. Antibodies against the following proteins were used: E-cadherin (24E10), vimentin (D21H3), p44/42 MAPK (Erk1/2), phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (D13.14.4E), GAPDH (D16H11) (from Cell Signaling Technology, Danvers, MA) and Ras [EP1125Y] (from Abcam, Cambridge, UK), followed by second antibody (zhongshanjinqiao, China). Protein bands were visualized using Super Enhanced Chemiluminescence Detection regents (Applygen Technologies, Beijing, China).

Cells were lysed in RIPA buffer (ThermoFisher Scientific) on ice for 15 min before centrifuging at 15,000 rpm for 15 min at 4 °C. Extracted proteins were quantified using the Pierce BCA assay (ThermoFisher Scientific) before loading 8 ug of total protein with 10% v/v LDS sample buffer (Life Technologies, Carlsbad, CA, USA) onto a 4–12% Bis-Tris pre-cast protein gel (Sigma-Aldrich). Samples were run at 150 V on ice for approximately 1 h before transferring to nitrocellulose membranes at 40 V for 1.5 h. The membranes were incubated in 5% skim milk for 1 h before incubation overnight at 4 °C in mouse anti-human LAMA5 (CL3118, Novus Biologicals, Centennial, CO, USA), mouse anti-human Hey2 (ab167280, ABCAM) or GAPDH (D16H11, Cell Signalling Technology, Danvers, MA, USA) diluted at 1:500 in 1% skim milk. After incubation in secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA), the membranes were incubated in ECL Prime solution (Amersham, Amersham Pl, Little Chalfont, Amersham, United Kingdon) and visualised using the Odyssey Fc imaging system (LI-COR Biosciences, Lincoln, NE, USA). Protein bands were quantified using ImageJ.