Protein estimation kit

Bovine serum albumin (BSA), ammonium sulfate, p-amino benzoic acid (PABA), p-nitro phenyl acetate (PNPA), trizma base (tris), iodo acetic acid, hexamethonium bromide, decamethonium bromide and protein estimation kit (Bradford Method) were obtained from Sigma. Phenyl methyl sulfonyl fluoride (USB, Switzerland) and DTT (SRL, India). All other chemicals and reagents were of analytical grade (Merck, India), unless or otherwise mentioned.

The extracellular vesicles were submitted to lysis using the RIPA buffer added with protease inhibitor cocktail. The resultant was analysed for total proteins using the protein estimation kit (Sigma Aldrich USA, Catlog# 51254). The proteins were incubated with specific antibodies overnight for obtaining blots. The antibodies used were specific for IL-6 (1:500) (ab6672), IL-1β (1:500) (ab9722), TNF-α (1:500) (ab6671), CD81 (1:1000) (ab109201), CD63 (1:1000) (ab231975), CD9 (1:1000) (ab223052) and HSP70 (1:1000) (ab2787), all the antibodies were obtained from Abcam USA. The blots were detected using Western blot detection system (Thermo Fisher USA).

Colorimetric assay was done for determining caspase-3 activity using a kit following the supplied instructions (SigmaAldrich USA). The N9 cells were isolated by centrifugation at 12,000 rpm for 10 min and then incubated in lysis buffer for 15 min. Further, the lysates were centrifuged at 12,000 rpm for 15 min, later the protein content was determined by protein estimation kit (SigmaAldrich USA) following the supplied instructions. The isolated lysates were incubated with Ac-DEVD-pNA (0.2 mM) (10 μl) reaction buffer for 2 h. The samples were evaluated using microplate reader at 405 nm.

After the chondrocytes were exposed to various concentrations of RT or AGEs the chondrocytes were rinsed with PBS and the cells were lysed using mixture of ice cold Radioimmunoprecipitation assay buffer (RIPA) buffer and phenyl-methanesulfonyl fluoride (1 mM), the cell lysate was obtained by centrifugation at 5000 rpm for 10 min at 4° C using a cooling centrifuge. The protein content was estimated using protein estimation kit (Sigma Aldrich USA). Protein (40 ng) was separated on sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and was then transferred to PVDF membranes. The membranes were blocked using non-fat milk (5%) for 2 hours and were incubated with I^ry antibodies for iNOS (1:500), p65 (1:500), COX-2 (1:500), IκBα (1 : 500), p-JNK (1 : 500), JNK (1 : 500), p-p38 (1:500), p38 (1:500), Lamin B1 (1 : 500) and GAPDH (1:500), TBST (2%) was used as diluent, the blots were incubated at 4° C for 12 hours. The blots were again exposed to II^ry antibodies for 2 hours at 25° C, the membranes were the rinsed with TBST and viewed by electro-chemiluminescence, the intensity of the blots was measured using Image pro software.

In order to establish the cross-reactivity of peptide₂₇ with the sera of VL-BT and healthy control group, indirect ELISA was performed following the published protocol as standardized in our laboratory (23 (link)) (detailed protocol has been included in the Supplementary Methods). The CICs were isolated from peripheral blood and subjected to protein A-mediated antigen dissociation (3 (link)). The protein content of the isolated CICs was estimated by employing bicinchoninic acid assay as well as Lowry's method (protein estimation kit; Merck Biosciences). Finally, sandwich ELISA was performed according to the protocol of Sengupta and coworkers (24 (link)). The detailed protocol experimental design has been provided in the Supplementary Material's methodology section.