2.4. Determination of protein expression of TLRs by Western blot

Since TLRs are primarily present in the membrane fraction, the protein expression of TLRs was determined by the Western blot method in membrane fractions, as described by us earlier (Dwivedi and Pandey, 2000).

Immunolabeling of TLRs was determined as described in a previous study (Dwivedi and Pandey, 2000). Briefly, equal volumes of membrane fractions isolated by this procedure (30 µg of protein in 20 µL) were loaded onto 7.5% (weight per volume) polyacrylamide gel and electrophoresed. The blots were initially developed using monoclonal anti-TLR2 (1:1000 dilution) and polyclonal anti-TLR1, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 antibody (1:1000 dilution) and subsequently using β-actin monoclonal antibody (1:5000 dilution). The levels of TLR1, TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 proteins were calculated as a ratio of the optical density of the TLR antibody of interest to the optical density of β-actin antibody. The antibodies for TLR1, TLR2, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10 were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, California, USA), and had molecular weights of 90 kDa, 100 kDa, 110 kDa, 96 kDa, 121 kDa, 120 kDa, 113 kDa and 90 kDa, respectively.