Journal: American Journal of Translational Research

Article Title: Proatherogenic stimuli induce HuR in atherosclerosis through MAPK/ErK pathway

doi:

Figure Lengend Snippet: HuR expression and its RNA-binding activity are upregulated in human and mouse atherosclerosis. (A) Western blot analysis of protein level of HuR in arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries (n = 5). Human Actin was used as a loading control. The representative images (left) and statistical analysis of band intensity (right) are shown. (B) qRT-PCR analysis of mRNA level of HuR in arterial extracts from non-atherosclerotic (n = 9) or atherosclerotic (n = 12) human coronary arteries. The results represent the mean value of 3 replicates and are normalized to human Actin. (C) Arterial extracts from non-atherosclerotic or atherosclerotic human coronary arteries were immunoprecipitated by HuR antibody or isotype IgG control antibody (n = 5). Total RNAs were purified from immunoprecipitates and reversely transcribed to cDNA and analyzed by qRT-PCR to measure the level of transcripts known to be associated with HuR, including COX-2, TNF-sure the level of transcripts known to be associated with than internal negative control. The results represent the mean value of 3 replicates. The enrichment value relative to IgG group for each transcript is normalized to human GAPDH. (D) Western blot analysis of protein level of HuR in HAECs and HASMCs isolated from arteries of 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE-/- mice (n = 5) fed a chow diet. VEGFR2, SM22 and Calponin were used as biomarkers. Representative images are shown. (E) qRT-PCR analysis of mRNA level of HuR in arterial extracts from 24-week-old C57BL/6 wild-type mice (n = 5) or ApoE-/- mice (n = 5) fed a chow diet. The results represent the mean value of 3 replicates and are normalized to mouse Actin. (F) As described in (C), arterial extracts from 24-week-old C57BL/6 wild-type mice or ApoE-/- mice fed a chow diet were subjected to in vitro RNA immunoprecipitation assay (n = 5). The enrichment value relative to IgG group for each transcript is further normalized to mouse GAPDH. The statistical analysis was performed using unpaired Student’s t-test. Data are mean ± s.e.m. **, P < 0.01; *, P < 0.05; NS, not significant.

Article Snippet: Reagents and antibodies The antibodies and reagents were purchased from the following sources: HuR (3A2) (Santa Cruz, sc-5261), SMCα-actin (Sigma, A2547), VEGFR2 (Cell Signaling, #2472), β-actin (AC-15) (Santa Cruz, sc-69879), SM22 (Santa Cruz, sc-373928), calponin (EP798Y) (abcam, ab46794), γ-Tubulin (GUT-88) (abcam, ab11316), goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004), goat anti-mouse IgG-HRP (Santa Cruz, sc-2302), goat anti-rat IgG-HRP (Millipore, AP136P); collagenase I (Sigma, C9891), collagenase II (Sigma, C6885) collagenase XI (Sigma, C7657), DNase I (Sigma, D5307), hyaluronidase (Sigma, H3506), recombinant IL1β (R&D System, NP-000567), recombinant TNF-α (R&D System, {"type":"entrez-protein","attrs":{"text":"P01375","term_id":"135934","term_text":"P01375"}} P01375 ), Ox-PAPC (InvivoGen, tlrl-oxp1); SB230580 (Sigma, S8307), PD98059 (Sigma, P215), SP600125 (Sigma, S5567), stattic (Sigma, S7947).

Techniques: Expressing, RNA Binding Assay, Activity Assay, Western Blot, Control, Quantitative RT-PCR, Immunoprecipitation, Purification, Negative Control, Isolation, In Vitro, RNA Immunoprecipitation

Journal: American Journal of Translational Research

Article Title: Proatherogenic stimuli induce HuR in atherosclerosis through MAPK/ErK pathway

doi:

Figure Lengend Snippet: Proatherogenic stimuli induce HuR expression and activity in vitro. (A) Western blot analysis of protein level of indicated proteins in cultured HAECs and HASMCs (n = 3). VEGFR2, SM22 and calponin were used as marker proteins. Human Actin was used as a loading control. The representative images (left) and statistical analysis of HuR band intensity (right) are shown. (B, C) Cultured HAECs were treated with vehicle or different concentrations of recombinant IL1β or TNF-α for 8 hours. (B) The mRNA level of HuR was measured by qRT-PCR analysis. The results represent the mean value of 3 replicates and are normalized to human Actin. (C) The protein level of HuR was measured by Western blot. Human Actin was used as a loading control and the representative images are shown. (D, E) Cultured HAECs were treated with vehicle control or different concentrations of Ox-PAPC for 8 or 12 hours. (D) The mRNA level and (E) protein level of HuR were determined as in (B, C). (F) HAECs were treated with vehicle control, 10 ng/ml recombinant IL1β or 50 µg/ml Ox-PAPC and cultured for 8 hours. Cell extracts were subjected to in vitro RNA immunoprecipitation assay as depicted in (Figure 1C and ​and1F).1F). The enrichment value relative to IgG group for each transcript is normalized to GAPDH. The statistical analysis was performed using unpaired Student’s t-test. Data are mean ± s.e.m. **, P < 0.01; *, P < 0.05; NS, not significant.

Article Snippet: Reagents and antibodies The antibodies and reagents were purchased from the following sources: HuR (3A2) (Santa Cruz, sc-5261), SMCα-actin (Sigma, A2547), VEGFR2 (Cell Signaling, #2472), β-actin (AC-15) (Santa Cruz, sc-69879), SM22 (Santa Cruz, sc-373928), calponin (EP798Y) (abcam, ab46794), γ-Tubulin (GUT-88) (abcam, ab11316), goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004), goat anti-mouse IgG-HRP (Santa Cruz, sc-2302), goat anti-rat IgG-HRP (Millipore, AP136P); collagenase I (Sigma, C9891), collagenase II (Sigma, C6885) collagenase XI (Sigma, C7657), DNase I (Sigma, D5307), hyaluronidase (Sigma, H3506), recombinant IL1β (R&D System, NP-000567), recombinant TNF-α (R&D System, {"type":"entrez-protein","attrs":{"text":"P01375","term_id":"135934","term_text":"P01375"}} P01375 ), Ox-PAPC (InvivoGen, tlrl-oxp1); SB230580 (Sigma, S8307), PD98059 (Sigma, P215), SP600125 (Sigma, S5567), stattic (Sigma, S7947).

Techniques: Expressing, Activity Assay, In Vitro, Western Blot, Cell Culture, Marker, Control, Recombinant, Quantitative RT-PCR, RNA Immunoprecipitation