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. 2022 Nov 19;25(12):105626. doi: 10.1016/j.isci.2022.105626

Figure 1.

Figure 1

Increased accumulation of unfolded protein in BRCA1-def cells, localization of BRCA1 protein to the ER, and interaction with ERAD E3 ligase complex components

(A) Photomicrographs of MDA-MB-436(+ or def) cells labeled with TPE-MI (light/blue) and concanavalin-A (red). Scale = 20 μm. TPE = TPE-MI. + = BRCA1-replete. def = BRCA1-deficient.

(B) Representative flow cytometry dot plots of TPE-high population in BRCA1-def and BRCA1+ cells.

(C and D) Quantitative analysis of TPE signal intensity of each cell preparation.

(E) Confocal microscopy images of BRCA1-proficient MDA-MB-231 breast cancer cells. Top, cells were immunostained with BRCA1 (red) and DERL1 (DERLIN-1) (yellow) antibodies. Bottom, cells were immunostained with secondary antibodies conjugated with fluorophores as control. All cells were counterstained with DAPI (blue). Scale = 5 μM. Green = GFP-KDEL.

(F and G) Immunoprecipitation (IP) of BRCA1 protein pull-downs of DERL1 and SEL1L from MCF7 (F) or MDA-MB-231 (G) cytoplasmic protein extract. IB = immunoblotting. IN = Input. Ig = non-specific immunoglobulin fragments. IgG(H) & IgG(L) = heavy and light chains. BRCA1 IB: ∗∗ = truncated BRCA1. ∗∗∗ = delta11q isoform. Data are represented as mean ± SD. P value was calculated by Student’s two-tailed, unpaired t-test. ∗∗∗ <0.001. See also Figures S1–S3.