sherbb3  (Addgene inc)

Journal: Oncogenesis

Article Title: Coordinate transcriptional regulation of ErbB2/3 by C-terminal binding protein 2 signals sensitivity to ErbB2 inhibition in pancreatic adenocarcinoma

doi: 10.1038/s41389-023-00498-8

Figure Lengend Snippet: A Immunoblot analysis of EGFR, ErbB2, ErbB3, CtBP2, and GAPDH (loading control) in lysates of CKP vs. CKP/KO and HPAC cells expressing control (shCtrl) or CtBP2-targeted (shCtBP2) shRNAs. B ErbB2/3 and CtBP2 mRNA levels were estimated by qPCR in CKP vs. CKP/KO and HPAC shCtrl vs. shCtBP2 cells. C (left panel) Lysates of shCtrl or shErbB3-expressing HPAC cells were immunoblotted with ErbB3 and GAPDH (loading control) antibodies. (right panel) The growth of shCtrl or shErbB3-expressing HPAC cells was monitored over 5 days using trypan blue staining to detect live cells as described . D Serum-starved CKP vs. CKP/KO cells were exposed to the indicated concentrations of recombinant mouse NRG-1 for 15 min and cell lysates were immunoblotted with the indicated antibodies. GAPDH was a loading control. n = 3 independent experiments. ** p < 0.01, *** p < 0.001 and **** p < 0.0001. p Values were obtained by performing two-tailed paired t -test between the two groups. Error bars indicate ±1.0 SD. Methods: (Generation of shRNA-expressing HPAC cell lines) HPAC (ATCC, Manassas, VA, USA) and all other human cells used in Fig. were grown in monolayers using DMEM media (Thermo Fisher) supplemented with 10% FBS (Corning) and 1% penicillin–streptomycin (Thermo Fisher) at 37 °C in 5% CO 2 . HPAC cell lines stably expressing control, CtBP2, or ErbB3-targeted shRNAs were derived by transduction of control, shCtBP2, or shErbB3-expressing lentiviruses prepared using the plasmids pLKO1-shCtrl (Cat No: SHC016; Sigma, St. Louis, MO, USA), pLKO.1-shCtBP2 (Sigma), and pLKO.1-ShErbB3 (Sigma) as described , followed by selection using puromycin (2 µg/ml) and verification of knockdown of intended shRNA targets by immunoblotting. (NRG-1 stimulation) CKP and CKP/KO cells were serum-starved overnight after reaching 80% confluency and treated with recombinant mouse NRG-1 (R&D Systems) at indicated concentrations for 15 min. Cells were then washed with ice-cold PBS, scraped on ice, and pelleted by cold centrifugation at 8000 rpm for 5 min at 4 °C. The cell pellet was lysed using cold 1× RIPA buffer (Cell Signaling Technology) containing protease and phosphatase inhibitors (Mini EDTA tablet, Roche) freshly added on ice and incubated for 10 min. Lysates were then centrifuged at 12,000 rpm for 10 min at 4 °C, protein concentration in the lysate was measured using BCA reagent (Thermo Fisher), and 30 µg of total protein was mixed with LDS buffer [1×] and heated for 10 min at 95 °C followed by immunoblotting. (Immunoblotting) Primary antibodies used for immunoblotting included: CtBP2 (Cat No: 612044, 1:1000; BD Biosciences), GAPDH (Cat No: AB 2302, 1:20000; MilliporeSigma), EGFR (Cat No: sc-03, 1:500; Santa Cruz Biotechnologies), Phospho-ErbB3 (Cat No: 4791, 1:1000; Cell Signaling Technology), ErbB3 (Cat No: 12708, 1:000; Cell Signaling Technology), Phospho-ErbB2 (Cat No: 2243, 1:1000; Cell Signaling Technology), ErbB2 (Cat No: sc-33684, 1:500; Santa Cruz Biotechnology), Total-Akt (Cat No: 9272, 1:1000; Cell Signaling Technology), Phospho-Akt (Cat No: 3787, 1:1000; Cell Signaling Technology), Total Erk1/2 (Cat No: 9102, 1:1000; Cell Signaling Technology), Phospho-Erk1/2 (Cat No: 4370, 1:1000; Cell Signaling Technology). After incubation in HRP-conjugated anti-rabbit or anti-mouse secondary antibodies (Cat Nos: 1706515 and 1706516, 1:10,000; Bio-Rad, Hercules, CA, USA), membranes were detected by chemiluminescence (Western Lightning Pro, Perkin-Elmer).

Article Snippet: HPAC cell lines stably expressing control, CtBP2, or ErbB3-targeted shRNAs were derived by transduction of control, shCtBP2, or shErbB3-expressing lentiviruses prepared using the plasmids pLKO1-shCtrl (Cat No: SHC016; Sigma, St. Louis, MO, USA), pLKO.1-shCtBP2 (Sigma), and pLKO.1-ShErbB3 (Sigma) as described [ ], followed by selection using puromycin (2 µg/ml) and verification of knockdown of intended shRNA targets by immunoblotting. (NRG-1 stimulation) CKP and CKP/KO cells were serum-starved overnight after reaching 80% confluency and treated with recombinant mouse NRG-1 (R&D Systems) at indicated concentrations for 15 min.

Techniques: Western Blot, Expressing, Staining, Recombinant, Two Tailed Test, shRNA, Stable Transfection, Derivative Assay, Transduction, Selection, Centrifugation, Incubation, Protein Concentration