405210 α rabbit hrp conjugated antibody cell signaling technology 7074 α gapdh polyclonal antibody  (Cell Signaling Technology Inc)

Journal: BMC Genomics

Article Title: Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages

doi: 10.1186/s12864-022-08612-7

Figure Lengend Snippet: FURIN and NFE2 expression after CRISPR edition. A FURIN RNA and protein expression. Cells were collected at T0 (before induction) and T3 (3 days after transdifferentiation induction). (CT0) and (CT3) negative control pDECKO-Intergenic at T0 and T3 respectively, (FUT0) and (FUT3) pDECKO-FURIN at T0 and T3, (FUT3s) pDECKO-FURIN at T3 and sorted from gate P4 (delayed population). Upper panel, qRT-PCR to check the expression of FURIN using two different sets of primers. Results are normalized to GAPDH and the fold change is calculated relative to the expression of cells infected with pDECKO-intergenic pgRNA at T3. The expression of FURIN decreases in cells infected with FURIN pgRNAs, especially in the delayed subpopulation (FUT3s). Bottom panel, western blot to assess the levels of the FURIN protein in BLaER1-Cas9 infected cells. Anti-FURIN antibodies recognize a band (marked with an arrowhead), the signal of which increases at T3, in line with RNA-Seq data (Supplementary Table S4). The FURIN band is not detectable in the pDECKO-FURIN infected cells (FUT3 and FUT3s). Uncropped blots are shown in Supplementary Fig. S A. B NFE2 RNA and protein expression. (CT0) and (CT2) negative control pDECKO-Intergenic at T0 (before induction) and T2 (2 days after transdifferentiation induction) respectively, (NFT0) and (NFT2) pDECKO-NFE2 at T0 and T2, (NFT2s) pDECKO-NFE2 at T2 and sorted from gate P4 (delayed population). Upper panel, qRT-PCR to check the expression of NFE2 using 2 different sets of primers. Results are normalized to GAPDH and the fold change is calculated relative to the expression of cells infected with pDECKO-intergenic T2. NFE2 expression in NFE2 pgRNA targeted cells is higher than in intergenic control cells (NFT2 and NFT2s compared to CT2). Bottom panel, western blot to check the protein levels of NFE2 in BLaER1-Cas9 infected cells. Anti-NFE2 antibodies detect two bands, the signal of which increases at T2 (CT2 compared to CT0). These two bands are strongly reduced in NFE2 targeted populations (NFT2 and NFT2s compared to CT2). Uncropped blots are shown in Supplementary Fig. S B

Article Snippet: As a protein loading control, the membranes were re-blotted with primary antibody rabbit anti-GAPDH-HRP polyclonal antibody (Proteintech, 10494–1-AP) 1:4,000 in blocking buffer, and incubated for 1 h at room temperature.

Techniques: Expressing, CRISPR, Negative Control, Quantitative RT-PCR, Infection, Western Blot, RNA Sequencing, Control