A cell line with target protein expression (HPA data) was selected for IF and FACS assays. Known/predicted subcellular localization of the target protein was also obtained from HPA or UniProt (table S4). For cell surface proteins, IF and FACS assays were performed under nonpermeable conditions without detergent in the buffers. For intracellular proteins, the permeable condition with 0.1% Triton added to the buffers was used throughout. Antibody binding signal was detected using Alexa Fluor 488 and 594 goat anti-mouse IgG secondary antibodies (Jackson ImmunoResearch, no. 115-545-003 and no. 115-585-003). Briefly, cells attached on coverslips (IF assays) or suspended in 1× PBS (FACS assays) were first fixed in 4% paraformaldehyde (PFA) for 10 min. PFA was then removed, and cells were rinsed three times with 1× PBS. Cells were blocked overnight at 4°C in blocking buffer (1× PBS containing 10% normal goat serum, 0.1% Triton was added for intracellular proteins). After removing the blocking buffer, cells were incubated with primary antibody (dilution in the blocking buffer at 1:100 to 1000) for 3 hours at room temperature. Cells were rinsed six times in 1× PBS before being incubated with fluorescence-labeled secondary antibody (diluted in blocking buffer at 1:500 dilution ratio with 1:10,000 Hoechst 33258; Sigma, no. 94403) for 1 hour. Last, cells were rinsed three times with 1× PBS. IF images were recorded with a Nikon confocal system A1Si. The three-dimensional reconstruction of the IF results was performed in ImageJ [National Center for Biotechnology Information, NIH (NCBI) free software]. IF staining patterns were compared with HPA data to confirm the subcellular localization of the target proteins. The FACS data were collected using a BD Accuri C6 Plus system. A control sample without primary antibody and another sample with isotype control antibody were used.

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