Figure 3.

Immunoprecipitation with the NCX1 (A; IP-NCX1) or CA IX (A; IP-CA IX) antibodies. Homogenates from the SiHa cells (normoxic (No), hypoxic (Hy) and hypoxic treated with KB-R7943 (HyKBR) for 48 h) were immunoprecipitated with NCX1 antibody or CA IX antibody, blotted onto PVDF membrane and subsequently incubated with CA IX/NCX1 and NHE1 antibodies. Data show that NHE1 co-precipitates with NCX1 as well as with CA IX (A). In SiHa DLD1 and A2780 cells, cytosolic Ca²⁺ was increased by hypoxic stimuli after 24 (B, gray columns) and also 48 h (B, black columns), while in hypoxic cells treated with KB-R7943 (HyKBR), cytosolic calcium was comparable to control, normoxic cells (B, No). Cytosolic sodium level was decreased due to 24 h hypoxia (C) and this decrease was prevented in the hypoxic SiHa cells treated with KB-R7943 (HyKBR). After 48 h of hypoxia, cytosolic sodium levels were comparable with those from normoxic cells (C). Results (B,C) are expressed as mean ± S.E.M. and represent an average from at least three independent measurements. Statistical significance * represents p < 0.05 and *** p < 0.001 compared to normoxic controls. Statistical significance ++ represents p < 0.01 and +++ p < 0.001 compared to hypoxic untreated cells.