Journal: Molecular pharmacology

Article Title: Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation.

doi: 10.1124/mol.64.4.811

Figure Lengend Snippet: Fig. 2. PTX down-regulates cyclin D1 expression and Rb phosphorylation in PDGF-stimulated mesangial cells. A, PTX down-regulates cyclin D1 mRNA expression. Growth-arrested cells were stimulated with PDGF in the presence or absence of PTX for various times. The levels of cyclin D1 and GAPDH mRNAs were determined by Northern blot analysis. B, PTX down-regulates cyclin D1 protein expression and Rb phosphorylation. Total cellular extracts were prepared from cells and then subjected to Western blot analysis with various antibodies to cyclin D1, Cdk4, p-Rb (Ser780), and -actin as indicated. Normalized signals from three inde- pendent experiments are shown in the bar charts as mean S.D. (C). *, p 0.01 versus growth-arrested cells; **, p 0.01 versus PDGF-stimu- lated cells in the absence of PTX.

Article Snippet: Antibodies against Thr308-phosphorylated Akt [p-Akt (Thr308)], Ser473-phosphorylated Akt [p-Akt (Ser473)], Ser780-phosphorylated retinoblastoma [p-Rb (Ser780)], Ser9-phosphorylated GSK3 (p-GSK3 ), and Ser 21/9-phosphorylated GSK3 / (p-GSK3 / ) were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Phospho-proteomics, Northern Blot, Western Blot

Journal: Molecular pharmacology

Article Title: Pentoxifylline inhibits platelet-derived growth factor-stimulated cyclin D1 expression in mesangial cells by blocking Akt membrane translocation.

doi: 10.1124/mol.64.4.811

Figure Lengend Snippet: Fig. 4. PDGF induces cyclin D1 expression in mesangial cells through PI3K/Akt. A and B, PTX inhibits cyclin D1 expression and Cdk4 activity induced by PDGF, compa- rable with the effect of LY294002. Growth-arrested mesangial cells were treated with or without PDGF, PTX (1 mM), and LY294002 (50 M) as indicated. Northern (A, top) and Western (A, bottom) blot analyses were performed as in Fig. 2. Cdk4 was immunoprecipi- tated from cell lysates and subjected to an in vitro kinase assay with GST-Rb as a substrate. The reaction mixtures were then used for Western blot analyses with an antibody to p-Rb (Ser780) (B). Normalized signals from three independent experiments are shown in the bar charts as mean S.D. (right). *, p 0.01 versus serum-deprived cells; **, p 0.01 versus PDGF-stimu- lated cells in the absence of PTX and LY294002. C, Akt is the downstream effector of PI3K in regulating cyclin D1 expression. Mesangial cells were transfected with either wild type (HA-Akt) or dominant-negative form (HA-Akt-K179A) of Akt, serum-starved for 24 h, and then stimulated with or without PDGF for 6 h. The expressions of cyclin D1 mRNA and protein were de- tected by Northern (top) and Western blot analyses (bottom). Normalized signals from four independent ex- periments are shown in the bar charts as mean S.D. (right). p 0.01 versus HA-Akt-transfected cells stim- ulated without (*) or with (**) PDGF.

Article Snippet: Antibodies against Thr308-phosphorylated Akt [p-Akt (Thr308)], Ser473-phosphorylated Akt [p-Akt (Ser473)], Ser780-phosphorylated retinoblastoma [p-Rb (Ser780)], Ser9-phosphorylated GSK3 (p-GSK3 ), and Ser 21/9-phosphorylated GSK3 / (p-GSK3 / ) were from Cell Signaling Technology (Beverly, MA).

Techniques: Expressing, Activity Assay, Northern Blot, Western Blot, In Vitro, Kinase Assay, Transfection, Dominant Negative Mutation

Journal: BMC complementary medicine and therapies

Article Title: Therapeutic efficacy of sorafenib and plant-derived phytochemicals in human colorectal cancer cells.

doi: 10.1186/s12906-023-04032-6

Figure Lengend Snippet: Fig. 13 Western blot analysis of the levels of cell cycle and apoptosis-associated proteins in the human colon cancer cell line SW1116 treated with Sora, Cur, and their simultaneous combinations. Cells were treated with Sora (5 µM), Cur (200 µM), Cur (400 µM) or a simultaneous combination of Sora (5 µM) and Cur (200 or 400 µM) for 72 h. The levels of proteins associated with cell cycle and apoptosis control were analysed using western blot analysis. A Cell cycle-associated proteins and (B) apoptosis-associated proteins. -actin was used as an internal control. Sora, sorafenib; Cur, curcumin; UT, untreated; pRb, phosphor retinoblastoma protein; Bcl-xL, Bcl extra-large protein. Full-length blots/gels are presented in Supplementary Fig. 1 Aa-Af, Ba-Be. Each protein was subjected to two separate western blot analyses. Because of the results’ similarity, the WB of every protein for each experiment has been merged into a composite figure and a representative figure was used for publication (Fig. 13 A, B)

Article Snippet: The membranes were washed three times for 5 min each with Tris-buffered saline containing 0.05% Tween-20 (TBST), and nonspecific binding sites were blocked by incubation with 5% bovine serum albumin-Tris-buffered saline Tween-20 buffer (BSA/ TBST; Bio-Rad Laboratories, Inc.) at 37 ̊C for 1 h. The membranes were subsequently washed again and incubated at 4 ̊C overnight with the following primary antibodies: Cyclin A2 rabbit mAb (E399; cat. no. ab32498), cyclin B1 XP® rabbit mAb (D5C10; cat. no. 12231), cyclin D1 rabbit mAb (92G2; cat. no. 2978), p27Kip1 XP® rabbit mAb (D69C12; cat. no. 3686), phosphorylated (p)-retinoblastoma protein (p-Rb) rabbit mAb (Ser780; C84F6; cat. no. 3590), cleaved-caspase-3 rabbit mAb (Asp175; 5A1E; cat. no. 9664), cleaved caspase-9 rabbit mAb (Asp330; D2D4; cat. no. 7237), Bax rabbit mAb (D2E11; cat. no. 5023), Bcl extra-large protein (Bcl-xL) rabbit mAb (54H6; cat. no. 2764) and β -actin rabbit mAb (cat. no. 4967; Cell Signalling Technologies, Inc.) at 1:1,000 dilutions in 5% BSA-TBST.

Techniques: Western Blot, Control