Extended Data Fig. 1: In vitro analysis of nanobody internalization.

(a) Median fluorescence intensity (MFI) of BMDMs treated with AlexaFluor647-labeled mouse serum albumin (MSA-AF647) at 1 µM or PBS in serum-containing ( + serum) or serum -deficient (-serum) media as measured by flow cytometry (n = 2). P values determined by ANOVA with post-hoc Tukey’s correction for multiple comparisons. (b) MFI of BMDM cells treated with 2 µM MSA-AF647 in serum containing ( + serum) or serum deficient (-serum) media as measured by flow cytometry (n = 2). P values determined by ANOVA with Šídák’s multiple comparison test. MFI of (c) BMDM cells, (d) BMDC cells, and (e) EMT6 cells treated with 1 µM nAlb-Cy5 or nGFP-Cy5 at 37 °C and 4 °C as measured by flow cytometry (n = 3). P values determined by two-tailed Student’s t-test. (f) MFI of EMT6 cells treated with 2 µM nAlb-Cy5 or nGFP-Cy5 in serum containing ( + serum) or serum deficient (-serum) media as measured by flow cytometry (n = 4). P values determined by ANOVA with Šídák’s multiple comparison test. (g-h) MFI of (g) EMT6, and (h) RAW 264.7 cells treated with nAlb-Cy5 (2 µM) with (+EIPA) or without (-EIPA) the macropinocytosis inhibitor EIPA as measured by flow cytometry (n = 3). P values determined by two-tailed Student’s t-test. (i) Integrated pixel intensity of Gal9-mCherry puncta per cell for cells treated DBCO-PEG₁₁-diABZI (DBCO-diABZI), nAlb-diABZI, and AP-diABZI at 0.25 µM (n = 9 for PBS; n = 3 for all other groups). P values determined via ANOVA with Dunnett’s multiple comparisons test for all groups vs. PBS; ns: not-significant (P > 0.05). (j-k) Colocalization analysis (j) of Cy5 and LysoTracker in RAW264.7 and EMT6 cells, and (k) fluorescent micrographs of RAW 264.7 cells treated with 2 μM nAlb-Cy5 or nGFP-Cy5 for analysis of colocalization of Cy5 (red) with lysosomes (LysoTracker Green; green); nuclei are stained with Hoechst (blue) (scale bar: 100 µm). Replicates are biological, and data are shown as mean ± SEM.