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. 2022 Nov 24;11:e76387. doi: 10.7554/eLife.76387

Figure 1. EROS stabilises the expression of gp91phox precursor.

(A–C) Mouse constructs encoding EROS and gp91phox were co-transfected into NIH3T3 (A), COS-7 (B), and HEK293T (C) cell lines. gp91phox expression was analysed by immunoblotting; arrow indicates gp91phox band; ns: non-specific band. (D–F) gp91phox and p22phox expression in HEK293T cells following transfection with the indicated human constructs. (G) Left panel: analysis of the stability of the different forms of gp91phox (indicated by the arrows) following transfection in HEK293T cells in the presence or absence of EROS and treatment with 10 μg/mL cycloheximide. Right panel: quantitation of the cycloheximide assay (mean of four independent experiments; error bars indicate SD) represented as a fold change of gp91phox in cells expressing gp91phox and EROS vectors relative to gp91phox vector alone at 0 hr and normalised to actin expression. Actin and vinculin were used as loading control. (H) Stability of endogenous gp91phox in PLB985 neutrophil-like cells overexpressing lentivirus (LV) EROS-GFP vector (MW ≈ 41 kDa) and treated with 10 μg/mL cycloheximide. (I–J) gp91phox expression following lentiviral transduction of EROS-GFP, gp91phox, or both in differentiated PL985 knockout (KO) for p22phox (I) or EROS (J). Data are representative of three independent experiments. See also Figure 1—figure supplement 1 and Figure 1—source data 1–4.

Figure 1—source data 1. Raw unedited blots for Figure 1A–C.
Figure 1—source data 2. Raw unedited blots for Figure 1D–F.
Figure 1—source data 3. Raw unedited blots for Figure 1G–J.
Figure 1—source data 4. Uncropped gels used for Figure 1A–J.

Figure 1.

Figure 1—figure supplement 1. EROS specifically regulates gp91phox not p22phox expression.

Figure 1—figure supplement 1.

(A) Expression of gp91phox in human induced pluripotent stem cells (iPS)-derived macrophage deficient for EROS (lanes are triplicate). (B) Diagram depicting the different stages of gp91phox biosynthesis and formation of the heterodimer with p22phox. (C) gp91phox expression upon co-expression of gp91phox-GFP and EROS-FLAG vectors compared to gp91phox GFP alone in non-adherent HEK293-F. (D) Abundance of the 58 kDa form of gp91phox in mouse NIH3T3 cells upon co-expression of gp91phox and EROS human constructs. (E) Expression of p22phox in HEK293 cells co-transfected with p22phox and EROS mouse constructs compared to p22phox vector alone (lanes are triplicate). (F) Expression level of EROS in bone marrow-derived macrophages (BMDM) from gp91phox knockout (KO) mice compared to control mice. Data are representative of three independent experiments. (G) Histogram derived from gp91phox stability curve (Figure 1H) in absence (blue) or presence (red) of EROS (four independent experiments; p-value was determined using unpaired Student’s t-test; error bars indicate SD). See also Figure 1—figure supplement 1—source data 1–3.
Figure 1—figure supplement 1—source data 1. Raw unedited blots for Figure 1—figure supplement 1A, C, and D.
Figure 1—figure supplement 1—source data 2. Raw unedited blots for Figure 1—figure supplement 1E and F.
Figure 1—figure supplement 1—source data 3. Uncropped gels used for Figure 1A, C–F.