Engineering of L-Plastin Peptide-Loaded Biodegradable Nanoparticles for Sustained Delivery and Suppression of Osteoclast Function In Vitro
Immunoblotting analyses with an L-plastin (LPL) and a p-Serine antibody. (a) Analysis of time-dependent expression of LPL in osteoclasts treated with (+; lanes 2-5) and without (-; lane 1) bone particles in the presence of TNF-α. (a) Immunoblotting analysis with an antibody to LPL (top panel) and GAPDH (bottom panel). (b) Amino acid sequences of TAT (11aa) -fused sNT-LPL (10aa) and control TAT alone (11aa) peptides are shown: P1) unsubstituted (S5S7); P2) double substituted (S5S7 to A5A7); P3) scrambled; P4) control TAT sequences only. (c). Immunoprecipitation and immunoblotting analyses. The effect of indicated sNT-LPL peptides (P1 to P4) on the phosphorylation of endogenous LPL is shown. An equal amount of osteoclast lysates was immunoprecipitated with an antibody to LPL and subjected to immunoblotting (IB) with a p-Serine antibody (top). This blot was stripped and blotted with an LPL antibody (panel (c); middle). Immunoprecipitation of lysates made from P2-transduced cells with a species-specific nonimmune serum was used as a control for immunoprecipitation (lane 5). An equal amount of total protein (Input) used for immunoprecipitation was assessed by direct immunoblotting of lysates with a GAPDH antibody. The experiment was repeated thrice and obtained comparable inhibitory effect with P1.