Recombinant Llama Anti-TMEM30A Single Domain Antibody (NABL-712CQ) (CAT#: NABL-712CQ)

Figure 1 Accumulation of FC5 antibody in the brain after i.v. injection into mice determined by optical imaging.

Quantification of the head region of interest average fluorescence concentration after injection of FC5 or NC11 or Cy5.5 alone.

Figure 2 Accumulation of FC5 antibody in the brain after i.v. injection into mice determined by optical imaging.

Quantification of the organs region of interest average fluorescence concentration after injection of FC5 or NC11 or Cy5.5 alone.

Figure 3 Effects of pharmacological inhibitors of adsorptive-mediated endocytosis (AME) and macropinocytosis on transmigration of FC5 across in vitro BBB model.

HCEC were pretreated for 30 minutes with AME inhibitors, protamine sulfate (40 μg/ml) and poly-1-lysine (300 μM), or micropinocytosis inhibitor, amiloride (500 μM), and FC5 transport was measured over 30 minutes as described in Materials and Methods.

Figure 4 Energy-dependence of FC5 uptake into HCEC and transmigration across in vitro blood-brain barrier model. Confocal microscopy images of FC5 uptake into HCEC at 37° C.

Transcellular migration of 10 μg/ml FC5 across HCEC at 37° C. or 4° C., or after a 30-min exposure of HCEC to 5 mM NaN3 and 5 mM deoxyglucose (2DG) for 20 min in glucose-free medium. FC5 transmigration was determined 30 min after addition to HCEC as described in Materials and Methods.

Figure 5 Energy-dependence of FC5 uptake into HCEC and transmigration across in vitro blood-brain barrier model. Confocal microscopy images of FC5 uptake into HCEC at 37° C.

The effect of Na+,K+-ATPase inhibitor, ouabain, on transcellular migration of FC5 across HCEC. Cells were pre-treated with 1 μM ouabain for 30 minutes and FC5 transport was measured over 30 minutes as described in Materials and Methods.

Figure 6 Role of clathrin-coated pits and caveolae in endocytosis and transcytosis of FC5 in HCEC.

Effects of pharmacological inhibitors of caveolae-mediated endocytosis, methyl-β-cyclodextrin (5 mM), nystatin (5 μg/ml) and filipin (5 μg/ml), or inhibitors of clathrin-coated pits-mediated endocytosis, chlorpromazine (50 μg/ml) or potassium-free buffer on transmigration of FC5 across in vitro BBB model.

Figure 7 Lack of transferrin receptor involvement in FC5 transcytosis across in vitro BBB.

Binding of the anti-transferrin receptor monoclonal antibody, CD71, FC5, pentameric construct of FC5 (P5) or non-related antibody from the same library that recognizes carbohydrate antigen, CEA, to human tranferrin receptor immobilized onto ELISA plate.