urea guanidine hydrochloride protein denaturing solution  (Ellman International Inc)

Journal: JACS Au

Article Title: Near-Infrared-Activated Photocages Made to Order: Late-Stage Caging Protocol

doi: 10.1021/jacsau.5c00223

Figure Lengend Snippet: (A) Caging of CDK4/6 inhibitor palbociclib in a single step by activated Cy7 photocage 16a , b , and subsequent uncaging by irradiation with 780 or 820 nm light. (B) Photouncaging of palbociclib from 8a in CD 3 OD by irradiation with 820 nm followed by 1 H NMR spectroscopy. (C) Mechanism of palbociclib actionbinding to CDK4/6 inhibits phosphorylation of Rb, leading to cell cycle arrest and death. (D) Photouncaging of palbociclib from 8b (12 μM) in PBS (10 mM, pH 7.4, 5% DMSO) followed by UV–vis spectroscopy (from magenta to cyan). (E) Kinetic traces at λ max of 8b (12 μM) in PBS (10 mM, pH 7.4, 5% DMSO) incubated in the dark (magenta) and irradiated at 820 nm (cyan). (F) Dose–response curves of 8b (left) and parent palbociclib (right) in the dark (magenta) or upon irradiation (cyan) with 780 nm on proliferation of MDA-MB-231 cancer cells (BrdU assay), n = 3. (G) Downregulation of Rb phosphorylation expressed as the percentage of pRB and RB with respect to vinculin remaining after 24 h of treatment in the dark (magenta) or after irradiation (cyan) with 780 nm light determined by Western Blot, n = 4. (H) Representative Western blots showing pRb, Rb and vinculin loading control 24 h after treatment with the 16b , 8b , or palbociclib (250 nM or 1 μM) in the dark and after the irradiation with 780 nm light, n = 3. (I) Immunofluorescence staining of Rb and pRb in cells treated with 8b , 16b , or palbociclib. (J) Immunofluorescence staining of ki67 (green) and pRb (red) in cells treated with 8b upon irradiation and in the dark. Means and standard deviations of the mean are given from at least 3 independent replicates. Scale bars represent 100 μm.

Article Snippet: Then, samples were fixed with the BrdU Fixative/Denaturating Solution (Millipore, Ja1598) and primary antibody Anti-BrdU 1:100 (Biolegend, 3D4) diluted in antibody diluent (Biolegend, Cat. 926001) was applied.

Techniques: Irradiation, Structural Proteomics, Phospho-proteomics, UV-Vis Spectroscopy, Incubation, BrdU Staining, Western Blot, Control, Immunofluorescence, Staining