Journal: Molecular Cancer Therapeutics

Article Title: Dual Targeting of CDK and Tropomyosin Receptor Kinase Families by the Oral Inhibitor PHA-848125, an Agent with Broad-Spectrum Antitumor Efficacy

doi: 10.1158/1535-7163.mct-10-0190

Figure Lengend Snippet: Figure 3. A, effect of PHA-848125 on phosphorylation status of pRb. A2780 cells were treated for 24 h with 1 and 3 μmol/L PHA-848125. Total cell lysates were immunoblotted and the different sites of phosphorylation were probed with specific antibodies. B, effect of PHA-848125 on several CDK-related proteins. A2780 cells were treated with 1 μmol/L PHA-848125 for indicated times. Total cell lysates were immunoblotted with specific antibodies. C, cell cycle distribution of A2780 cells treated with 1 μmol/L PHA-848125 was determined by flow cytometry at different time points and compared with the corresponding control cells. D, the same samples used for cell cycle evaluation were analyzed by immunocytochemical staining for BrdUrd incorporation to quantify DNA replication. E, cell cycle distribution of A2780 cells, untreated or treated with 1 and 3 μmol/L PHA-848125 for 24 h. F, effect of PHA-848125 on TRKA phosphorylation and downstream pathway. DU-145 cells were serum starved and pretreated with different doses of the compound for 30 min before NGF stimulation for 15 min. Total cell lysates were immunoblotted with specific antibodies.

Article Snippet: Immunoblotting was done according to ard procedures, and staining was done with the folg antibodies against: retinoblastoma protein (pRb), B1, cyclin D1, cyclin A, and Kip/p27 (Pharmingenosciences); Cdc6 (NeoMarkers); p53, Cdc25A, TRKA, hospho-TRKA (Santa Cruz Biotechnology); hospho-Thr821 and pRb-phospho-Ser249/252 (Bio); and pRb-phospho-Ser780, pRb-phospho-Ser807/811, ho-phospholipase Cγ, phospho-AKT, and total (Cell Signaling).

Techniques: Phospho-proteomics, Flow Cytometry, Control, Staining

Journal: ACS chemical neuroscience

Article Title: Small molecules targeting PTPσ—Trk interactions promote sympathetic nerve regeneration

doi: 10.1021/acschemneuro.1c00854

Figure Lengend Snippet: Representative western blots (left) probing for PTPσ and TrkA following TrkA-RFP immunoprecipitation. HEK-293t cells were transfected with TrkA-RFP and PTPσ, treated with either vehicle (DMSO), (A) HJ-01, (B) HJ-02, or (C) HJ-03 then stimulated with CSPGs and NGF. Quantification (right) of PTPσ that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. HJ-01 and HJ-02 reduced the amount of PTPσ that bound to TrkA-RFP at 1μM, but HJ-03 had no effect. (D) HEK-293t cells were transfected with TrkA-RFP and p75NTR, treated with vehicle (DMSO) and HJ-02, then stimulated with CSPGs and NGF. Quantification (right) of p75NTR that co-immunoprecipitated with TrkA-RFP normalized to vehicle treated cells. Data are mean ± SD, n=5-9 experiments; n.s.- not significant, *p<.05, **p<.01 vs. vehicle treated control, one-way ANOVA (Dunnett multiple comparisons post-test).

Article Snippet: We would like to thank Dr. Moses Chao (Skirball Institute, NYU) for the HEK-TrkB cells and for the TrkA-RFP and p75NTR plasmids (Addgene plasmid # 24093, 24091).

Techniques: Western Blot, Immunoprecipitation, Transfection