Nsc23766

d-Pantethine, imipramine, GW4869, calpeptin, Y-27632, imatinib mesylate, sulfisoxazole, bisindolymaleimide I, indomethacin, NSC23766, clopidogrel, glibenclamide, Chloramidine, amiloride, and U0126 were purchased from Selleckchem (Selleckchem, Houston, TX, USA). Manumycin A and cytochalasin D were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Luis, MO, USA). All compounds (purity > 97%) were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Luis, MO, USA), at a concentration of 50 mM, before use.

The Rac1 inhibitor, NSC23766 (Selleckchem, S8031), was administered as described previously [37 (link), 38 (link)]. For in vivo studies, 4.0 mg/kg NSC23766 or the vehicle control was administered intraperitoneally once every two days following the AAV2-shPorf-2 injection. For in vitro experiments, 30 μM NSC23766 or the vehicle was added to the retinal explant medium, and the medium was changed once every two days. The detailed culture methods for retinal explants are described above.

The primary antibodies used in Western blot analysis were purchased from the following commercial suppliers: Dock4 (ab85723, 1:1000) and PSD95 (ab2723, 1:1000) were from Abcam; GluA1 (PC246, 1:1000), GluA2 (MAB397, 1:1000), GluN2A (AB1555P, 1:1000), GluN2B (AB1557P and 06-000, 1:1000), Rac1 (05-389, 1:2000), and puromycin (MABE343, 1:10000) were from Merck Millipore; GluN1 (32-0500, 1:1000) was from Invitrogen; Dock4 (WH0009732M1, 1:1000), ɑ-tubulin (T6199, 1:5000), and Flag (F1804, 1:1000) were from Sigma; GAPDH (A01020, 1:5000) was from Abbkine. The secondary antibodies for Western blot were purchased from Cell Signaling Technology (anti-mouse IgG-HRP, 7076s; anti-Rb IgG-HRP, 7074s). Pharmacological inhibitors MG132 and NSC23766 were purchased from Selleck. puromycin was purchased from Merck Millipore. cDNAs of Dock4 and Rac1 and their mutants, and Dock4 shRNA were described previously [27 (link), 32 (link)]. Neuro-2a cells were purchased from ATCC and were routinely tested for mycoplasma contamination-free before use.

Polypeptide (GeneScript China, Nanjing, Jiangsu, China) was synthesized by use of FlexPeptide^TM technology. The sequences are LSTAADMQGVVTDGMASGLDKDYLKPDDAWVLGEA (P1) and LSTAADMQGVVTDGMASGLDKDYLKPDDAVWLGEA (P2). FITC conjugated P1 was provided by GeneScript China as well. Polypeptide powder was resolved in PBS to a final concentration of 1.0 μM before use and PBS served as control. NSC23766 (Selleck Chemicals, Houston, TX, USA) was resolved in H₂O and used at a final concentration of 100 μM in cell cultures.

All cells were cultured at 37°; air; 95%; CO₂, 5%. H358 (NCI-H358), H23 (NCI-H23), H1792 (NCI-H1792) and 293T cells were obtained from American Type Tissue Culture (ATCC). Cells were regularly checked for mycoplasma contamination by polymerase chain reaction^{64 (link)} and found to be negative. H358 sgRB1#4 parental and resistant cell lines were verified by STR profiling (Labcorp, Burlington, NC, USA). LUAD cells were grown in RPMI−1640 medium (Gibco, 11875119) supplemented with 10% fetal bovine serum (FBS) (Gibco, 12483020) and 1% Pen/Strep (Gibco, 15140-122). 293T cells were grown in DMEM medium complete with 10% FBS and 1% Pen/Strep (Gibco, 15140-122). For cells and experiments with doxycycline-inducible constructs, cells were grown in RPMI-1640 medium (Gibco, 11875119) supplemented with 10% tetracycline-free FBS (Clontech, 631101) and Pen/Strep (Gibco, 15140-122). Doxycycline hyclate (Sigma-Aldrich, D9891) was added to cells at 200 ng/mL when indicated. Trametinib (Selleckchem, S2673), SCH772984 (Selleckchem, S7101), AMG 510 (Selleckchem, S8830), SB 747651 A (Tocris, 4630), MK-2206 (Selleckchem, S1078), NSC 23766 (Selleckchem, S8031), dabrafenib (Selleckchem, S8031), infigratinib (Selleckchem, S2183) and N-acetyl-L-cysteine (Sigma-Aldrich, A7250) were added to cells when indicated. Experiments were performed on cells between passages 4-20.

Antibodies for PAK1, phospho-PAK1 and phospho-Ser/Thr were from Cell Signaling Technology; IFT88, KIF3A, SuFu, c-Myc, Gli1, acetylated-αTubulin (Ac-Tub), Vav2, GAPDH and β-actin antibdies were from Santa Cruz Biotechnology; Ptch1 antibody, Smo antibody and phospho-Vav2 antibody were from Abcam; Gli2 antibody and Rac1 antibody were from Bioss (Beijing, China); Rac2 and Rac3 antibodies were purchased from Huabio (Hangzhou, China); Flag-tag and HA-tag antibodies were from Beyotime (Shanghai, China); GST-tag antibody was from Yeason (Shanghai, China), and antibody anti-Arl13b was from Proteintech. Alexa555- and Alexa-488 conjugated secondary antibodies were from Life Technology. Recombinant mouse Shh protein (N-Shh, C25II, N-Terminus) was from R&D Systems (#464-SH). Cyclopamine, SAG and NSC23766 were purchased from Selleckchem. Cycloheximide and MG132 were from Beyotime (Shanghai, China).