Journal: Carcinogenesis

Article Title: The lung-enriched p53 mutants V157F and R158L/P regulate a gain of function transcriptome in lung cancer

doi: 10.1093/carcin/bgz087

Figure Lengend Snippet: uPA protein expression is mutp53-dependent. Protein expression of uPA encoded by the RNA-seq identified gene PLAU, was assessed in p53-depleted and control conditions. (A) Western blot of A549, H2087, H441, H2110 and H2009 cells transfected with negative control or siRNA directed against p53 (25 nM). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is shown as a loading control. (B) qRT–PCR was performed to evaluate mRNA expression of PLAU after depletion of p53 by siRNA. mRNA expression values are normalized to GAPDH and siCtrl condition within each cell line. (C) Protein and conditioned media were harvested from cells after overnight incubation in reduced-serum conditions. Nitrocellulose membranes were stained with Ponceau S dye to visualize protein bands for loading control. Protein expression of pro-uPA (49 kDa) and its cleaved and activated forms B-chain (33 kDa) and A-chain (19 kDa) are shown in cell lysates and conditioned media. (D) Protein and E. mRNA expression of uPA (PLAU) following depletion of V157F mutp53 by siRNA.

Article Snippet: The following antibodies were used to detect protein expression by western blot: p53 (DO-1, sc-126; Santa Cruz), p21 (C-19, sc-397, Santa Cruz) and uPA (AF1310, R&D systems).

Techniques: Expressing, RNA Sequencing, Control, Western Blot, Transfection, Negative Control, Quantitative RT-PCR, Incubation, Staining

Journal: Journal of the American Society of Nephrology

Article Title: Sphingomyelinase-Like Phosphodiesterase 3b Expression Levels Determine Podocyte Injury Phenotypes in Glomerular Disease

doi: 10.1681/asn.2013111213

Figure Lengend Snippet: Figure 5. Interaction of suPAR and SMPDL3b. (A) Co-IP experiments performed in HEK293 cells showing an interaction between GFP- SMPDL3b and FLAG-uPAR (F-uPAR; upper panel) but not between GFP-SMPDL3b and FLAG-b3 integrin (F-b3; lower panel). FLAG- empty vector (F-C) was used as a negative control (upper panel). (B) Endogenous immunoprecipitation showing interaction of SMPDL3b and suPAR in glomeruli isolated from mice injected with PBS or LPS. E1, eluate from PBS-injected mice; E2, eluate from LPS-injected mice; I1, input (glomerular lysate) from PBS-injected mice; I2, input (glomerular lysate) from LPS-injected mice; IP, immunoprecipitation; WB, Western blot. (C) Competitive Co-IP experiments performed in HEK293 show that increasing amounts of GFP-SMPDL3b interfere with the interaction of FLAG-uPAR/suPAR and GFP-b3 integrin. However, transfection of GFP-empty vector used as a control does not affect the interaction between uPAR/suPAR and b3 integrin. GFP, green fluorescent protein.

Article Snippet: The biopsy tissue specimens were manually microdissected,41–43 and glomerular gene expression profiling was performed as previously described.15 Measurements of suPAR Circulating suPAR levels were determined in the sera from 53 patients with FSGS from the FSGS clinical trial25 and 30 patients with type 1 diabetes and normoalbuminuria, 34 patients with type 1 diabetes and microalbuminuria, and 10 patients with type 1 diabetes and macroalbuminuria from the FinnDiane study cohort using Quantikine Human uPAR Immunoassay (R&D Sys- tems) performed following the manufacturer’s protocol.

Techniques: Co-Immunoprecipitation Assay, Plasmid Preparation, Negative Control, Immunoprecipitation, Isolation, Injection, Western Blot, Transfection, Control