Hi, I am trying to see heat shock induced HSP70 expression in N2A cells. I have tried heat shock at 42 deg celsius and 43 deg celsius for 30 mins and 1.5 hours respectively followed by recovery at 37 deg celsius for 2 hours and 4.5 hours respectively.

After lysis and western blotting, an intense band below 75kDa is detected by the antibody MAB16631 (R&D systems), which does not increase in intensity even after heat shock.

Are there other factors I should be aware of while giving heat shock to cells, to see HSP70 induction under heat stress?