mGluR1 expression and silencing. <i>GRM1</i> overexpression results in mGluR1 protein overexpression in MCF10AT1 cells.

<p><b>A. <i>GRM1</i> expression.</b> RNA lysates from stable transfectants of <i>GRM1</i>-MCF10AT1 or control <i>LACZ</i> cells were isolated and subjected to QPCR as described in Materials/Subjects and Methods. <i>GRM1</i> was expressed at a level 1.6×10⁵ times higher in stable transformed GRM1-MCF10AT1 cells than in <i>LacZ</i> control infected cells. <b>B. mGluR1 expression.</b> Protein lysate (30 µg) was loaded in each lane. The membrane was stripped and reprobed for tubulin antibody to show equal loadings. (<b><i>LacZ</i></b> = pLenti-LacZ; <b><i>GRM1</i></b> = pLenti-<i>GRM1</i>). <b>C, D. </b><b><i>GRM1</i></b><b> silencing.</b> RNA lysates from MCF10DCIS.com and MCF10CA1d cells with Lentivirus-driven expression of sh<i>GRM1</i> or NS vector were subjected to qPCR analysis. <i>GRM1</i> expression was significantly silenced by 0.5 and 0.25 fold in sh<i>GRM1</i> transduced MCF10DCIS.com and MCF10CA1d cells respectively, compared to the NS control infected cells (<b>C</b>). GRM1-MCF10AT1 cells were silenced with either sh<i>GRM1</i> or NS control vector and <i>GRM1</i> mRNA message was analyzed using QPCR. <i>GRM1</i> is significantly silenced by 0.48 fold in <i>GRM1-</i>MCF10AT1 cells compared to the NS control infected cells (<b>D</b>). <b>E. mGluR1 silencing.</b> Protein lysate (50 µg) was loaded in each lane. mGluR1 is also silenced by sh<i>GRM1</i> in <i>GRM1</i>-MCF10AT1 cells compared to the NS control cells. qPCR was performed in triplicate and results are recorded as the mean ± SEM of three experiments, where *p<0.05 compared to control. (<b>For C, D, and E: NS</b> = pLenti vector containing nonsilencing control: <b>sh</b><b><i>GRM1</i></b> = pLenti vector containing shRNA against <i>GRM1</i>.)