Mast cell expression and HDAC expression on IgE exposure:
Mast cell were grown to confluence in six well cell culture
plates exposed to 10ng/ml & 20 ng/ml concentration of IgE for 16
hours in serum free medium. Cells were then collected and centrifuged at
3000g, washed in 1X-PBS, lysed using RIPA buffer. The lysate after
protein estimation (Pierce™ BCA Protein Assay Kit Cat No: 23225, MA,
USA) was used for western blot analysis (30 μg/μl) to assess endogenous
Histone Deacetylase (HDAC) expression using Antibody Sampler Kit
(#9928, Cell signalling Technology, MA, USA) using the corresponding
antibodies namely, HDAC1, HDAC2, HDAC4, HDAC6 and 7 (representing Zinc
dependent class I, class IIa and IIb ) as well as the Beta actin
(β-Actin Antibody (AC-15) sc-69879, Santa Cruz Biotechnology, CA, USA)
used as the loading control. Chemidoc-XRS+ (Biorad, CA, USA) was
used for gel documentation and image lab tool (Biorad, CA, USA) was used
for estimating the pixel density of the bands.