enolase 1 antibody  (Bio-Techne corporation)

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

doi: 10.1016/j.matbio.2018.08.003

Figure Lengend Snippet: A, B) Measurement of HuR protein by Western blot (A) and corresponding densitometric analysis (B) confirmed successful knockdown of HuR after transduction with HuR-directed shRNA under normoxia and hypoxia. C) Both cytoplasmic levels of HuR significantly decreased following NP cell transduction with shHuR. D) Proliferation potential of HuR-silenced and control NP cells under hypoxia up to 72 h. E, F) HuR silencing exhibited no effects on select HuR/HIF-1α transcriptional targets - Hif-1α, Phd2, Phd3, Pdk1, Pgk1, Pfkfb3, Vegf, Eno1, Ldhb under both normoxia and hypoxia. Western blot images shown in A and C are from one representative experiment performed in ≥ 3 biological replicates. Quantitative data are represented as mean ± SD of at least three independent experiments. ***, p ≤ 0.001; NS, not significant; Cy, Cytoplasmic; Nu, Nuclear; Nx, Normoxia; Hx, Hypoxia.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in TBST and incubated overnight at 4 o C in blocking buffer with anti-HuR (1:400, Santa Cruz, sc-5261); anti-HIF-1α (1:500, R&D Systems, MAB1536); anti-PHD2 (1:1000, Cell Signaling Technology, 4835); anti-PHD3 (1:1000, Novus Biologicals, NB100–139); anti-PDK1 (1:1000, Novus Biologicals, NBP1–85955); anti-PGK1 (1:1000, Abcam, ab137575); anti-PFKFB3 (1:1000, Abcam, ab181861); anti-ENO1 (1:1000, Novus Biologicals, NB100–65252); anti-SDC4 (1:1000, EMD Millipore, ABT157); anti-MMP3 (1:2000, Abcam, ab52915);anti-MMP13 (1:3000, Abcam, ab39012); anti-CA12 (1:1000, Cell Signaling Technology, 5865); anti-lamin A/C (1:1000, Cell Signaling Technology, 2032); or anti-β-tubulin (1:3000, DSHB, E-7).

Techniques: Western Blot, Transduction, shRNA

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

doi: 10.1016/j.matbio.2018.08.003

Figure Lengend Snippet: A, B) Heat map and volcano plot depicting differentially expressed genes between control and HuR knockdown NP cells. C) RNA sequencing of HuR-silenced NP cells confirmed decreased expression of HuR (Elavl1), along with significant changes in positively regulated (Bcl2, Idh1) and negatively regulated (Cdkn1b) known HuR target genes. Log2(fold change) values are shown. D) RNA sequencing of HuR-silenced NP cells showed no significant changes of select HuR/HIF-1α transcriptional targets - Hif-1α, Phd3, Pdk1, Pgk1, Pfkfb3, Vegf, Eno1, Ldhb. E, F) Log2(fold change) values for matrix-related transcripts that were differentially expressed between control and HuR knockdown NP cells. NS, not significant.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in TBST and incubated overnight at 4 o C in blocking buffer with anti-HuR (1:400, Santa Cruz, sc-5261); anti-HIF-1α (1:500, R&D Systems, MAB1536); anti-PHD2 (1:1000, Cell Signaling Technology, 4835); anti-PHD3 (1:1000, Novus Biologicals, NB100–139); anti-PDK1 (1:1000, Novus Biologicals, NBP1–85955); anti-PGK1 (1:1000, Abcam, ab137575); anti-PFKFB3 (1:1000, Abcam, ab181861); anti-ENO1 (1:1000, Novus Biologicals, NB100–65252); anti-SDC4 (1:1000, EMD Millipore, ABT157); anti-MMP3 (1:2000, Abcam, ab52915);anti-MMP13 (1:3000, Abcam, ab39012); anti-CA12 (1:1000, Cell Signaling Technology, 5865); anti-lamin A/C (1:1000, Cell Signaling Technology, 2032); or anti-β-tubulin (1:3000, DSHB, E-7).

Techniques: RNA Sequencing Assay, Expressing

Journal: Matrix biology : journal of the International Society for Matrix Biology

Article Title: RNA binding protein HuR regulates extracellular matrix gene expression and pH homeostasis independent of controlling HIF-1α signaling in nucleus pulposus cells

doi: 10.1016/j.matbio.2018.08.003

Figure Lengend Snippet: A) Schematic showing the workflow of the Messenger ribonucleoprotein immunoprecipitation (mRNP-IP or RIP) assay. B) Validation of cell lysates and post-IP samples from HuR RIP assay. HuR was immunoprecipitated (IP) using a rabbit polyclonal antibody, and IP was validated by a mouse monoclonal antibody by Western blot analysis. β-Tubulin was used as a loading control for the input and a negative control for the post-IP samples. Lamin A/C was used as a control to detect nuclear contamination in the input. C-E) The relative abundance of Tgfb3, Sdc4, Hif-1α, Vegf, Col1a1, Pgk1, Pdk1, Eno1, Pfkfb3, Acan and Ca12 mRNA bound to HuR, normalized to IgG isotype controls, was determined by qRT-PCR using 18S rRNA as a loading control. Gapdh was used as a negative control and HuR as a positive control. Western blot images shown in B are from one representative experiment out of 4 independent experiments. Quantitative data are represented as Mean ± SD. *, p ≤ 0.05; **, p ≤ 0.01; NS, not significant.

Article Snippet: Membranes were blocked with 5% non-fat dry milk in TBST and incubated overnight at 4 o C in blocking buffer with anti-HuR (1:400, Santa Cruz, sc-5261); anti-HIF-1α (1:500, R&D Systems, MAB1536); anti-PHD2 (1:1000, Cell Signaling Technology, 4835); anti-PHD3 (1:1000, Novus Biologicals, NB100–139); anti-PDK1 (1:1000, Novus Biologicals, NBP1–85955); anti-PGK1 (1:1000, Abcam, ab137575); anti-PFKFB3 (1:1000, Abcam, ab181861); anti-ENO1 (1:1000, Novus Biologicals, NB100–65252); anti-SDC4 (1:1000, EMD Millipore, ABT157); anti-MMP3 (1:2000, Abcam, ab52915);anti-MMP13 (1:3000, Abcam, ab39012); anti-CA12 (1:1000, Cell Signaling Technology, 5865); anti-lamin A/C (1:1000, Cell Signaling Technology, 2032); or anti-β-tubulin (1:3000, DSHB, E-7).

Techniques: Immunoprecipitation, Western Blot, Negative Control, Quantitative RT-PCR, Positive Control