Figure S4.
CRISPR/Cas9 gene editing IRAK4 or IRAK1 gene loci with mScarlet and analysis of MyD88-GFP and IRAK4/1-mScarlet colocalization.(A) Schematic of the IRAK4 (top) and IRAK1 (bottom) gene locus and HDR template designed to insert a mScarlet open reading frame immediately upstream of the stop codon. EL4 cells were electroporated with HDR and gRNA/Cas9 plasmids to simultaneously edit MyD88 and IRAK4 or IRAK1 gene loci. Dual edited cells were selected by FACS and PCR (see Fig. S1, A–D for workflow, and Materials and methods). (B) Western blot analysis of three MyD88-GFP/IRAK4-mScarlet–expressing EL4 clones. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of fluorescent protein open reading frames at both gene loci. All data presented in the manuscript were acquired with clone 3. (C) Western blot analysis of MyD88-GFP/IRAK1-mScarlet–expressing EL4 clone. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of mScarlet or mEGFP open reading frames at both gene loci. See Figs. S7 and S8 for uncropped blots. (D–G) Data shown from clonal MyD88-GFP/IRAK4-mScarlet–expressing EL4 cells (green, D and E) or IRAK1-mScarlet (orange, F and G). (D and F) Data points are the proportion of individual cells from independent experiments. Bars are experimental replicate means. (E and G) Vertical line in E and G is at 4.5× MyD88-GFP. (D) Percentage (%) of MyD88-GFP puncta that colocalizes with IRAK4-mScarlet, combined, and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Violin plot of the percent of MyD88-GFP puncta that is colocalized with IRAK4-mScarlet, combined, and categorized by lifetime (<50 s or ≥50 s). Few tracks recruit IRAK4 (“All”, n = cells, replicates 1–3: 2.4%, n = 30; 3.2%, n = 31; 3.3%, n = 30). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 1.0%, n = 30; 1.2%, n = 31; 1.1%, n = 30). However, MyD88-GFP puncta that persist for ≥50 s are more likely to recruit IRAK4 (“≥50 s”, n = cells, replicates 1–3: 28%, n = 30; 35%, n = 31; 32%, n = 30). (E) Maximum MyD88-GFP size of IRAK4-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (green) or noncolocalized (black) with IRAK4-GFP. MyD88-GFP puncta colocalized with IRAK4-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 11× MyD88-GFP, n = 835 versus 3.1× MyD88-GFP, n = 29,072; 10× MyD88-GFP, n = 601 versus 3.3× MyD88-GFP, n = 17,221; 11.7× MyD88-GFP, n = 552 versus 3.3× MyD88-GFP, n = 17,854). (F) Percentage (%) of MyD88-GFP puncta that colocalize with IRAK1-mScarlet, combined and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Few tracks recruit IRAK1 (“All”, n = cells, replicates 1–3: 6.3%, n = 15; 1.5%, n = 32; 7.6%, n = 40). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 3.9%, n = 15; 0.74%, n = 32; 4.1%, n = 40). However, MyD88 puncta that persist for ≥50 s are more likely to recruit IRAK1 (“≥50 s”, n = cells, replicates 1–3: 41%, n = 15; 9.0%, n = 32; 59%, n = 40). (G) Maximum MyD88-GFP size of IRAK1-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (orange) or not colocalized (black) with IRAK1-GFP. MyD88-GFP puncta colocalized with IRAK1-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 19× MyD88-GFP, n = 291 versus 5.6× MyD88-GFP, n = 5,401; 6.2× MyD88-GFP, n = 314 versus 2.4× MyD88-GFP, n = 14,125; 10× MyD88-GFP, n = 1,439 versus 3.1× MyD88-GFP, n = 16,834). -ve, negative; +ve, positive; MW, molecular weight.

CRISPR/Cas9 gene editing IRAK4 or IRAK1 gene loci with mScarlet and analysis of MyD88-GFP and IRAK4/1-mScarlet colocalization. (A) Schematic of the IRAK4 (top) and IRAK1 (bottom) gene locus and HDR template designed to insert a mScarlet open reading frame immediately upstream of the stop codon. EL4 cells were electroporated with HDR and gRNA/Cas9 plasmids to simultaneously edit MyD88 and IRAK4 or IRAK1 gene loci. Dual edited cells were selected by FACS and PCR (see Fig. S1, A–D for workflow, and Materials and methods). (B) Western blot analysis of three MyD88-GFP/IRAK4-mScarlet–expressing EL4 clones. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of fluorescent protein open reading frames at both gene loci. All data presented in the manuscript were acquired with clone 3. (C) Western blot analysis of MyD88-GFP/IRAK1-mScarlet–expressing EL4 clone. Lysates were probed with anti-IRAK4, anti-RFP, anti-MyD88, and anti-GFP to confirm editing and insertion of mScarlet or mEGFP open reading frames at both gene loci. See Figs. S7 and S8 for uncropped blots. (D–G) Data shown from clonal MyD88-GFP/IRAK4-mScarlet–expressing EL4 cells (green, D and E) or IRAK1-mScarlet (orange, F and G). (D and F) Data points are the proportion of individual cells from independent experiments. Bars are experimental replicate means. (E and G) Vertical line in E and G is at 4.5× MyD88-GFP. (D) Percentage (%) of MyD88-GFP puncta that colocalizes with IRAK4-mScarlet, combined, and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Violin plot of the percent of MyD88-GFP puncta that is colocalized with IRAK4-mScarlet, combined, and categorized by lifetime (<50 s or ≥50 s). Few tracks recruit IRAK4 (“All”, n = cells, replicates 1–3: 2.4%, n = 30; 3.2%, n = 31; 3.3%, n = 30). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 1.0%, n = 30; 1.2%, n = 31; 1.1%, n = 30). However, MyD88-GFP puncta that persist for ≥50 s are more likely to recruit IRAK4 (“≥50 s”, n = cells, replicates 1–3: 28%, n = 30; 35%, n = 31; 32%, n = 30). (E) Maximum MyD88-GFP size of IRAK4-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (green) or noncolocalized (black) with IRAK4-GFP. MyD88-GFP puncta colocalized with IRAK4-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 11× MyD88-GFP, n = 835 versus 3.1× MyD88-GFP, n = 29,072; 10× MyD88-GFP, n = 601 versus 3.3× MyD88-GFP, n = 17,221; 11.7× MyD88-GFP, n = 552 versus 3.3× MyD88-GFP, n = 17,854). (F) Percentage (%) of MyD88-GFP puncta that colocalize with IRAK1-mScarlet, combined and by MyD88-GFP lifetime (<50 s or ≥50 s) per cell across experimental replicates. Few tracks recruit IRAK1 (“All”, n = cells, replicates 1–3: 6.3%, n = 15; 1.5%, n = 32; 7.6%, n = 40). It is especially evident in MyD88-GFP puncta that persist for <50 s (“<50 s”, n = cells, replicates 1–3: 3.9%, n = 15; 0.74%, n = 32; 4.1%, n = 40). However, MyD88 puncta that persist for ≥50 s are more likely to recruit IRAK1 (“≥50 s”, n = cells, replicates 1–3: 41%, n = 15; 9.0%, n = 32; 59%, n = 40). (G) Maximum MyD88-GFP size of IRAK1-mScarlet colocalized and noncolocalized puncta across experimental replicates. Density plot of MyD88-GFP size categorized as colocalized (orange) or not colocalized (black) with IRAK1-GFP. MyD88-GFP puncta colocalized with IRAK1-mScarlet are brighter (mean colocalized versus not colocalized, n = puncta, replicates 1–3: 19× MyD88-GFP, n = 291 versus 5.6× MyD88-GFP, n = 5,401; 6.2× MyD88-GFP, n = 314 versus 2.4× MyD88-GFP, n = 14,125; 10× MyD88-GFP, n = 1,439 versus 3.1× MyD88-GFP, n = 16,834). -ve, negative; +ve, positive; MW, molecular weight.

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