ANO8 increases native and expressed STIM1-Orai1 complexes and the number and size of ER/PM junctions.Figure 2(A) FRET ratio was measured with HEK cells transfected with STIM1-CFP, Orai1-YFP (black) and co-transfected with untagged ANO8 (red) before and after store depletion with 25 µM CPA in Ca2+-free solution. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. TIRF microscopy was used to quantify puncta formation at the TIRF field in cells transfected with STIM1-CFP, ANO8-YFP and Orai1-mCherry before and after store depletion. (A) Example images. (B) Time course of STIM1 puncta formation in cells transfected with STIM1 and Orai1 alone (black) or together with ANO8 (red). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (C-E) Effect of ANO8 on the rate of STIM1-STIM1 clustering (C), number of STIM1 puncta (D) and number of Orai1 puncta (E). The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (F) HEK cells were used to immunoprecipitate (IP) the native ANO8 (A8) or Orai1 (O1) and blotted (B) for STIM1. R=resting, S=stimulated. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (G) HEK cells treated with scrambled (siScr) or siANO8 (siA8) were used to IP the native Orai1 (O1) or STIM1 (S1) and blotted (B) for the native STIM1, Orai1 or ANO8, as indicated. The columns show the mean±s.e.m of 4 independent experiments. The results are mean±s.e.m and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (H) Co-IP of myc-STIM1 and Orai1-HA, myc-STIM1 and ANO8-YFP, and Orai1-HA and ANO8-YFP was measured in transfected HEK cells before (R, resting) and in response to store depletion (S, stimulated). Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (I) Effect of ANO8-YFP on myc-STIM1 and Orai1-HA at the ER/PM junction. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (J-L) Junctional Orai1 (J), STIM1 (K) and ANO8 (L) in resting (R) and store-depleted cells (S). The results are mean±s.e.m of 4 experiments and difference were analyzed by unpaired t test. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (M) Example EM images recorded from HEK cells transfected with empty vector (left) or ANO8 (right). Red arrows mark ER/PM junctions. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed. (N, O) The size (N) and density (O) of ER/PM junctions in vector-transfected (control) and ANO8-transfected cells. Data information: The first number in parenthesis indicates the number of similar experiments performed and the second number is the number of cells analyzed. All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed.