Journal: Molecular Medicine

Article Title: Interferons and IRF-1 Induce Expression of the Survival Motor Neuron (SMN) Genes

doi: 10.1007/bf03401830

Figure Lengend Snippet: Fig. 1. Induction of survival motor neuron gene (SMN) expression in interferon (IFN)- treated cells. (A) HeLa cell proteins (50 g) were solubilized and run on a 12% polyacrylamide gel and blotted. The Western blot was cut into two strips and each strip was probed with (a) the preim- mune serum or (b) the immune serum (739785) at a dilution 1:2000. (B) 24 hr after subculture, exponen- tially growing A172 and HOG cells were treated with or control without 1000 U/ml of IFN- or 1000 U/ml of IFN-. After 16 hr of culture, cells were lysed and total protein extracts (50 g) were analyzed by Western blots analysis, using the polyclonal anti- body against SMN (739785) (1:2000) followed by horseradish (HRP)-labeled conjugate antibody at a dilution 1:2000. Immunodetection was realized by enhanced chemiluminescent protein (ECL) reagents

Article Snippet: The human astrocytoma cell line A172 (CRL1620) was obtained from the American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Expressing, Western Blot, Stripping Membranes, Control, Labeling, Immunodetection

Journal: Molecular Medicine

Article Title: Interferons and IRF-1 Induce Expression of the Survival Motor Neuron (SMN) Genes

doi: 10.1007/bf03401830

Figure Lengend Snippet: Fig. 4. Interferon regulatory factor (IRF)-1 partic- ipates in interferon (IFN)-induced expression of SMN and SMNc genes. (A) Nuclear extracts pre- pared as described in “Materials and Methods’’ from untreated (lanes 1, 5), IFN--treated (lanes 2–4), or IFN--treated (lanes 6–8) were assayed for specific binding activity to an oligonucleotide probe corre- sponding to SMN fragment 2 224 to 2 202 (9), after a 30 min preincubation without (lanes 1, 2, 5, 6) or with rabbit affinity-purified polyclonal antibodies against human IRF-1 (lanes 3, 7) or signal transduc- ers and activators of transcription (Stat1; lanes 4, 8). Arrows indicate C1-, C2-, and C3-specific protein- DNA complexes in IFN--treated cells and C4-, C5-, and C6-specific protein-DNA complexes in IFN- -treated cells. (B) Total RNA from A172 cells treated for 4 hr and 8 hr, with medium alone (control; C) or in the presence of IFN- (1000 U/ml) or IFN- (1000 U/ml) was reverse-transcribed and polymerase chain reaction (PCR) coamplification was performed for IRF-1 and -actin genes, as described in “Materi- als and Methods.” The products were analyzed on a 2% agarose gel and visualized by ethidium bromide staining.

Article Snippet: The human astrocytoma cell line A172 (CRL1620) was obtained from the American Type Culture Collection (ATCC; Rockville, MD).

Techniques: Expressing, Binding Assay, Activity Assay, Control, Reverse Transcription, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Intestinal Lipid Handling

doi: 10.1161/atvbaha.113.302993

Figure Lengend Snippet: Figure 4. Intestinal lipid synthesis and lipoprotein biogenesis in the small intestine of insulin-sensitive and insulin-resistant obese subjects. Tissue homogenates were analyzed by immunoblotting for the phosphorylation of 5′-adenosine monophosphate–activated protein kinase α (AMPKα) at Thr172 (A), AMPKα (B), acetyl-CoA carboxylase (ACC) at Ser79 (C), and ACC (D). Densitometric analyses of protein expres- sion were normalized for protein expression levels of β-actin. Intestinal de novo lipogenesis rates were measured by the incorporation of [1-14C]-acetic acid for 3 h (E). Data are expressed as nanomoles of acetic acid incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Apolipoprotein (Apo) B-48 synthesis by intestinal explants was evaluated by the incorporation of [35S]-methionine in immunopurified Apo B-48 resolved on SDS-PAGE acrylamide gel (F). Data are expressed as DPM of [35S]-methionine incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Triglyceride (TG)-rich lipoprotein (TRL) production by intestinal explants was evaluated by the incorporation of [1-14C]-oleic acid in TRL isolated by ultracentrifugation (G). Data are expressed as DPM of [1-14C]-oleic acid incorporated by a milligram of protein (insulin-sensitive, n=7; insulin-resistant, n=9). Data are mean±SEM (*P<0.05).

Article Snippet: The following antibodies (directed against human) and dilutions (1:1000 unless otherwise specified) were employed: mouse anti-β-actin (1:40000, Sigma Aldrich, St. Louis, USA); anti-Akt Veilleux et al., Data Supplements, ATVB/2013/302993D, Page 2 (#ab32902) and anti-TNF-α (#ab66579) from Abcam (Cambridge, USA); antiintestinal-fatty acid binding protein (I-FABP) and anti-liver-fatty acid binding protein (L-FABP) antibodies were raised in rabbits after injection of recombinant proteins;3 anti-microsomal transfer protein (MTP) was kindly provided by John Wetterau and Harris Jamil (Bristol-Myers Squibb Research Institute, USA);4 antiSAR-1B (1:2000) was kindly provided by Randy Schekman (University of California, USA);4 anti-PCSK9 was kindly provided by Geneviève Dubuc and Jean Davignon (Clinical Research Institute of Montreal, Canada);5 anti-nuclear factor kappa B (NF-kB) p65 subunit (sc-372G) and anti-I-KappaB-alpha (IKB-α) (sc-1643) were obtained from Santa-Cruz Biotechnology (Santa-Cruz, USA); anti-phospho-Akt Ser473 (#9271), anti-phospho-AMPKα Thr172 (#40H9), antiAMPKα (#2532), anti-phospho-p38 MAPK Thr180/Tyr182 (#4631), anti-p38 MAPK (#9212), anti-phospho-JNK Thr183/Tyr185 (#9251), anti-JNK (#9252), anti-phospho-Acetyl-CoA Carboxylase (ACC) Ser79 (#3661) and anti-ACC (#3662) were obtained from Cell Signaling Technology (Boston, USA).

Techniques: Western Blot, Phospho-proteomics, Expressing, SDS Page, Acrylamide Gel Assay, Isolation