Figure 2: Validation of ERβ antibodies on positive and negative control cells.
From: Insufficient antibody validation challenges oestrogen receptor beta research
(a) Representative images of IHC staining pattern on control cells. Left panel: ERβ antibodies PPZ0506 (Invitrogen, dilution 1:250), 14C8 (GeneTex, 1:1,500) and PPG5/10 (DAKO, 1:60) on ERβ-positive (HCT116-ERβ, top) and ERβ-negative (HCT116-Mock, bottom) control cells. Right panel: ERα antibody 1D5 (DAKO, 1:150) on ERα-positive (T47D-Mock, top) and ERα-negative (HCT116-Mock, bottom) control cells. Scale bar in top left image indicates 50 μm. (b) Representative images of western blotting on control cells. Left panels: Indicated ERβ antibodies (dilution 1:1,000, except for PPG5/10 which is 1:200) on recombinant ERβ (rERβ), ERβ-negative cell lysate (HCT116-Mock), and ERβ-positive cell lysate (HCT116-ERβ), performed on the same cell lysates run one gel on consecutive lanes. Right panel: ERα antibody 1D5 (1:1,000) on ERα-negative (HCT116-Mock) and ERα-positive (T47D-Mock) control cells. Lower panels show loading control (beta-actin). (c) Summary of proteins detected by MS in IPs of the respective antibodies in 50–80 kDa gel bands in replicated experiments. Blue font indicates expected antibody-targeted protein, purple non-intended targets, and grey proposed general binders.
