Journal: Cancer discovery

Article Title: PTEN -null Tumors Cohabiting the Same Lung Display Differential Akt Activation and Sensitivity to Dietary Restriction

doi: 10.1158/2159-8290.CD-12-0507

Figure Lengend Snippet: A, Representative pictures of KPTENp53 tumor cell colonies (10× magnification). B, SPC, PTEN and GAPDH protein levels in KPTENp53 cells compared to NSCLC PC-9 cells. C, N-glycosylation levels in bronchiolar KPTENp53 cell lines with stable knockdown of control GFP (shGFP) or ENTPD5 (shE5-1), as assessed by PHA-E lectin blotting. GAPDH protein levels are represented as a loading control. D, Levels of ENTPD5, IGF1Rβ, phospho-S473 Akt, total Akt and GAPDH in shGFP and shE5-1 cells grown for 24 hours in media supplemented with 10% serum (S) or with different concentrations of IGF1 (25 or 50 ng/ml) or insulin (10, 50 or 100 ng/ml), in the absence of serum. E, ENTPD5, IGF1Rβ, phospho-S473 Akt, total Akt, phospho-T24/T32 FOXO1/3a (p-FOXO1/3a, low and high exposure levels), total FOXO1, BiP, CHOP, cleaved capase-3 (casp-3) and GAPDH protein levels in shGFP and shE5-1 cells described in (C), grown in the presence of 10%, 3% or 1.5% serum for 72 hours. F, Graphs representing quantified changes (obtained by ImageJ) in p-Akt/T-Akt, p-FOXO1/T-FOXO1, or ENTPD5, IGF1Rβ, BiP, CHOP cleaved caspase-3, over GAPDH protein levels assayed for in (E). (C-F) are representative of at least three independent experiments. In (D) and (E), separated lanes of individual blots were acquired from a single electrophoresis gel.

Article Snippet: For N-glycosylation assessment, protein lysates were resolved by 10% SDS-PAGE and analyzed by blotting using HRP-conjugated PHA-E lectin (USBiological, #P3370-25).

Techniques: Glycoproteomics, Knockdown, Control, Electrophoresis

Journal: Nucleic Acids Research

Article Title: Regulation of human Dicer by the resident ER membrane protein CLIMP-63

doi: 10.1093/nar/gks903

Figure Lengend Snippet: Dicer transits through the ER of cultured human cells and is glycosylated. ( A ) 293T-REx cells, treated (+) or not (−) with doxycyclin (Dox) to induce shRNA-mediated downregulation of CLIMP-63 expression, were exposed to Brefeldin A for 18 h. Cells were harvested and protein extracts were analyzed by immunoblotting using anti-Dicer (upper panel) and anti-CLIMP-63 (lower panel) antibodies. ( B ) Dicer immunoprecipitates prepared from 293T-REx cells were analyzed by immunoblotting using HRP-conjugated PHA-E (left panel), a lectin that can recognize complex oligosaccharide structures or anti-Dicer antibody (right panel). Asterisk symbol indicates the expected glycosylated Dicer protein band. ( C ) Immune complexes from 293T-REx cells conditionnally expressing short hairpin RNA directed against CLIMP-63 mRNA (shCLIMP-63-1) following doxycyclin treatment (Dox) for 6 days were analyzed as in (B). NSA, non-specific antibody.

Article Snippet: Protein extracts were analyzed by western blot using the following primary antibodies: rabbit polyclonal anti-Dicer ( , ), mouse monoclonal anti-Dicer (Abcam), mouse monoclonal anti-CLIMP-63 (Alexis Biochemicals), mouse monoclonal anti-HA (Roche), mouse monoclonal anti-actin AC-40 (Sigma), rabbit polyclonal anti-calreticulin (Affinity Bioreagents), rabbit polyclonal anti-Drosha (Upstate Cell Signalling Solutions), rabbit polyclonal anti-Coactosin-like protein (CLP) ( ) and horseradish peroxidase (HRP)-conjugated Phaseolus vulgaris E (PHA-E) (USBiological).

Techniques: Cell Culture, shRNA, Expressing, Western Blot