Figure S3.
Differential expression of the A1aY1 binder stably incorporated in U2OS cells.(A) Blue sensor (TagBFP A1aY1). (B) Red sensor (TagRFP-T A1aY1). Upper and lower panels show representative images of cells expressing low-medium and high levels of A1aY1 fused to either TagBFP or TagRFP-T, respectively. Scale bars = 10 µm. (C and D) Representative image of FACS of 10,000 nontransfected cells (C, marked in red) and transfected cells with N-terminal 6xHis TagRFP-T_A1aY1 (D, marked in purple and blue). The cells were sorted into two bins, low/medium (in purple) and high expression (blue), based on the fluorescence intensity of TagRFP-T. The left panels in C and D represent the forward- versus side-scattered plot of the cell population considered for sorting and marked as P1 population. (E and F) Western blot images for the estimation of the N terminus 6xHis TagRFP-T_A1aY1 (6xHis tag red tyrosination sensor) and total tubulin levels. A total of 10 µg protein lysate was loaded for each low/medium- and high-level expression FACS sorted cell lysate samples (Materials and methods). Purified 6xHis-A1aY1-GFP and goat brain tubulin of known concentrations were loaded to estimate the concentration of the sensor and tubulin in the lysate. Blots were probed with anti-hexahistidine (E) and mouse monoclonal DM1A antibody (F) for detection of 6xHis-A1aY1-GFP and tubulin samples, respectively. The expected bands for the sensor and the tubulin is marked in red box on top of the blot. The sensor and the recombinant purified A1aY1-GFP migrates as two bands in the SDS gel. (G and H) Scatter plots for the gel band intensities versus concentrations of purified 6xHis-A1aY1-GFP (G) and goat brain tubulin (H). The titration curve was used to estimate the concentration of sensor and total tubulin in the cell lysate as shown in I. (I) Table comparing the concentration of the sensor in FACS sorted cell lysates from low-medium and high-level expression of the N-terminal 6xHis TagRFP-T_A1aY1. * and ** denote the low/medium- and high-expression cell samples and is expressed as ∼1.5- and ∼3-fold excess compared with the total tubulin levels in the cells, respectively. A.U., arbitrary units.

Differential expression of the A1aY1 binder stably incorporated in U2OS cells. (A) Blue sensor (TagBFP A1aY1). (B) Red sensor (TagRFP-T A1aY1). Upper and lower panels show representative images of cells expressing low-medium and high levels of A1aY1 fused to either TagBFP or TagRFP-T, respectively. Scale bars = 10 µm. (C and D) Representative image of FACS of 10,000 nontransfected cells (C, marked in red) and transfected cells with N-terminal 6xHis TagRFP-T_A1aY1 (D, marked in purple and blue). The cells were sorted into two bins, low/medium (in purple) and high expression (blue), based on the fluorescence intensity of TagRFP-T. The left panels in C and D represent the forward- versus side-scattered plot of the cell population considered for sorting and marked as P1 population. (E and F) Western blot images for the estimation of the N terminus 6xHis TagRFP-T_A1aY1 (6xHis tag red tyrosination sensor) and total tubulin levels. A total of 10 µg protein lysate was loaded for each low/medium- and high-level expression FACS sorted cell lysate samples (Materials and methods). Purified 6xHis-A1aY1-GFP and goat brain tubulin of known concentrations were loaded to estimate the concentration of the sensor and tubulin in the lysate. Blots were probed with anti-hexahistidine (E) and mouse monoclonal DM1A antibody (F) for detection of 6xHis-A1aY1-GFP and tubulin samples, respectively. The expected bands for the sensor and the tubulin is marked in red box on top of the blot. The sensor and the recombinant purified A1aY1-GFP migrates as two bands in the SDS gel. (G and H) Scatter plots for the gel band intensities versus concentrations of purified 6xHis-A1aY1-GFP (G) and goat brain tubulin (H). The titration curve was used to estimate the concentration of sensor and total tubulin in the cell lysate as shown in I. (I) Table comparing the concentration of the sensor in FACS sorted cell lysates from low-medium and high-level expression of the N-terminal 6xHis TagRFP-T_A1aY1. * and ** denote the low/medium- and high-expression cell samples and is expressed as ∼1.5- and ∼3-fold excess compared with the total tubulin levels in the cells, respectively. A.U., arbitrary units.

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