mouse cnot6l  (Santa Cruz Biotechnology)

Figure Legend Snippet: Fig. 2. IL-8 mRNA is a target of CNOT6L. A. IL-8 mRNA expression in proliferating (P) and differentiating LHCN human myoblasts. IL-8 mRNA amount was normalized to cyclophilinA and expressed as a relative amount to P condition. Results are presented as the mean ± SD of triplicates. B. Quantification of IL-8 mRNA bound by CNOT6L in C2C12 cells. Following 2 days of differentiation, relative IL8 mRNA levels were quantified by RT-qPCR in FH-CNOT6L transfected cells and in control cells after FLAG-HA immunoprecipitation. IL-8 amount was normalized to total RNA and expressed as relative amount to pRev condition. Results are expressed as the mean ± SD of 4 experiments. Lower panel: representative WB with HA antibody for eluted exogenous CNOT6L after FLAG-HA immunoprecipitation. C. Stability of IL-8 mRNA. LHCN cells were transfected with CNOT6L-targeting siRNA (no. 1 or 2) or a control siRNA at day 0. Cells were placed in differentiation conditions for 48 h and treated with DRB (5, 6-Dichlorobenzimidazole riboside) for the indicated amount of time. IL-8 mRNA was quantified as described in A and reported as a relative expression to T0. Results are expressed as the mean ± SD of 3 independent experiments performed in duplicates. D. Poly(A) tail length assay of IL-8 mRNA. LHCN cells were transfected with CNOT6L-targeting siRNA or a control siRNA, placed in differentiation conditions and total mRNAs were extracted. IL-8 coding sequence (CS) was amplified by RT-PCR and IL-8 CS amplicon amount (normalized to that of CyclophilinA) was used to adjust the quantity of IL-8 mRNA used in PAT assay. Poly(A) tail length of IL-8 mRNA was evaluated by RT-PCR using 2 different primers (1 or 2) and size of minimum PCR product is indicated. Right panel: representative pattern on 2% agarose gel. Statistical significance was evaluated by bilateral unpaired two-sample Student's t-test. ⁎⁎⁎p b 0001, ⁎⁎p b 0.01, ⁎p b 0.05.

Techniques Used: Expressing, Quantitative RT-PCR, Transfection, Control, Immunoprecipitation, Sequencing, Reverse Transcription Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

Figure Legend Snippet: Fig. 3. IL-8, a novel CNOT6L target, controls muscle cell differentiation. A. IL-8 protein expression in supernatant of proliferating (P) and differentiating LHCN human myoblasts. IL-8 protein amount (μg/mL) was quantitated by ELISA in the supernatants of LHCN cell cultures over the course of differentiation. Results are expressed as the relative amount of IL-8 compared to P condition (mean ± SD of three independent replicates). B–C. Functional role of IL-8 on LHCN differentiation by gain- or loss-of-function assays. Differentiation of LHCN was induced with or without recombinant IL-8 (3 μg/mL) for gain-of-function assay (B) or with IL-8 neutralizing or isotypic antibodies at the indicated concentrations for loss-of-function assay (C). Differen- tiation of LHCN was monitored quantifying the number of myotubes at 5 (B) or 7 (C) days of differentiation as described in Fig. 1D. Left panels: typical acquisition fields. Right panels: Data are expressed as the mean ± SD of 3 independent experiments performed in triplicates. D. IL-8 protein level in CNOT6L KD LHCN cell culture supernatants. LHCN cells were transfected with a CNOT6L-targeting siRNA (no. 1 or 2) or a control siRNA. After 3 days of differentiation, IL-8 protein level was quantified by ELISA. Results are expressed as the mean ± SD of two independent experiments performed in triplicates. E. LHCN cells were transfected with CNOT6L-targeting siRNA or a control siRNA. Differentiation was assayed as described in B–C, with neutralizing IL-8 or isotypic antibody. Data are expressed as the mean ± SD of 3 independent experiments performed in triplicates. Statistical significance was evaluated by bilateral un- paired two-sample Student's t-test ⁎⁎⁎p b 0001, ⁎⁎p b 0.01, ⁎p b 0.05.

Techniques Used: Cell Differentiation, Expressing, Enzyme-linked Immunosorbent Assay, Functional Assay, Recombinant, Cell Culture, Transfection, Control