Journal: Molecular Cell

Article Title: Conformational surveillance of Orai1 by a rhomboid intramembrane protease prevents inappropriate CRAC channel activation

doi: 10.1016/j.molcel.2021.10.025

Figure Lengend Snippet:

Article Snippet: The following antibodies were used for western blotting (WB) and immunofluorescence (IF): mouse anti-beta-actin (Santa Cruz, catalog number sc-47778; WB 1:2000), mouse anti-HA (ENZO, catalog number ENZ-ABS120-0200; WB 1:1000), mouse anti-transferrin receptor (Invitrogen, catalog number 13–6800; WB 1:1000), rabbit anti-Stim1 (Cell Signaling Technology, catalog number 5668S (D88E10); WB 1:2000), rabbit anti-Orai1 (Sigma Aldrich, catalog number O8264; WB 1:2500), goat anti-myc tag (Abcam, catalog number ab9132; IF 1:2000), rabbit anti-RHBDL2 (Proteintech, catalog number 12467-1-AP; WB 1:250 – only detected RHBDL2 in HaCaT lysates), rabbit anti-V5 tag (Cell Signaling Technology, catalog number 13202S; WB and IF 1:2000).

Techniques: Virus, Recombinant, Gene Expression, Control, Negative Control, shRNA, Mutagenesis, Plasmid Preparation, Software

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 and stromal-interacting molecule 1 (STIM1) expression in mouse pulmonary arterial smooth muscle cells (PASMCs). A: RT-PCR products from cultured mouse PASMCs amplified using primers for mouse Orai1 (269 bp), STIM1 (473 bp), and β-actin (498 bp). Three separate RT-PCR reactions were performed in the presence (+) and absence (−) of reverse transcriptase (RT). B: Orai1, STIM1, and GAPDH proteins were detected in cultured mouse PASMCs using Western blot analysis. Experiments were performed in 5 separate Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Amplification, Reverse Transcription, Western Blot

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: STIM1 is associated with Orai1 to mediate CCE in mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in nontransfected mouse PASMCs and in PASMCs transfected with 200 nM scrambled siRNA (negative control). The expression of STIM1 but not Orai1 or GAPDH was reduced significantly in cells transfected with 200 nM STIM1 siRNA. The expressions of STIM1 and Orai1 but not GAPDH were reduced significantly in cells transfected with both 200 nM STIM1 siRNA and 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: siRNA knockdown of STIM1 reduced the CPA-induced transient and sustained increase in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the CPA-induced transient but not sustained increase in fura-2 fluorescence ratio in the presence of nifedipine. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in negative control cells (filled bars, n = 103), in STIM1 siRNA-transfected cells (shaded bars, n = 148), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bars, n = 70). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with negative control cells and STIM1 siRNA-transfected cells (ANOVA). E: siRNA knockdown of STIM1 reduced the increase in Mn2+ quench of fura-2 fluorescence caused by 10 μM CPA in the presence of 10 μM nifedipine. siRNA knockdown of STIM1 and Orai1 further reduced the increase in Mn2+ quench of fura-2 fluorescence caused by CPA in the presence of nifedipine. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in negative control cells (filled bar, n = 125), in STIM1 siRNA-transfected cells (shaded bar, n = 151), and in STIM1 siRNA and Orai1 siRNA-transfected cells (open bar, n = 137). **P < 0.01, compared with negative control cells (ANOVA); ++P < 0.01, compared with the negative control cells and STIM1 siRNA-transfected cells (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Transfection, Negative Control, Expressing, Western Blot, Knockdown, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: knockdown of Orai1 reduced CCE in STIM1-overexpressing mouse PASMCs. A and B: STIM1, Orai1, and GAPDH proteins were detected in cells infected with adenovirus containing green fluorescent protein (Ad-GFP) transfected with 200 nM scrambled siRNA. The expression of STIM1 but not Orai1 or GAPDH increased markedly in cells infected with STIM1-GFP-adenovirus (Ad-GFP-STIM1) transfected with scrambled siRNA. The expression of Orai1 but not STIM1 or GAPDH was reduced significantly in STIM1-overexpressing cells transfected with 200 nM Orai1 siRNA. Experiments were performed in 3 separate Western blot analyses (**P < 0.01, ANOVA). C: overexpression of STIM1 in scrambled siRNA-transfected cells caused an increase in CPA-induced transient and sustained rise in fura-2 fluorescence ratio in the presence of 10 μM nifedipine. The increases in fluorescence ratio were reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. D: bar graph showing mean changes in transient and sustained increase in [Ca2+]i caused by 10 μM CPA after readdition of 2 mM Ca2+ in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 33), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 65), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 50). *P < 0.05, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA). E: overexpression of STIM1 in scrambled siRNA-transfected cells caused a CPA-induced increase in Mn2+ quench of fura-2 fluorescence in the presence of 10 μM nifedipine. The increases in Mn2+ quench of fura-2 fluorescence was reduced in Orai1 siRNA-transfected cells overexpressed with STIM1. F: bar graph showing percentage change in fura-2 quench rate after store-depletion in the presence of 10 μM nifedipine, in GFP-infected cells transfected with scrambled siRNA (filled bars, n = 40), in STIM1-overexpressing cells transfected with scrambled siRNA (shaded bars, n = 95), and in STIM1-overexpressing cells transfected with Orai1 siRNA (open bars, n = 57). **P < 0.01, compared with GFP-infected cells transfected with scrambled siRNA and STIM1-overexpressing cells transfected with Orai1 siRNA (ANOVA).

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Knockdown, Infection, Transfection, Expressing, Western Blot, Over Expression, Fluorescence

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Orai1 coimmunoprecipitates with STIM1 in mouse PASMCs. A, left: Orai1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of Orai1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. B, left: STIM1 was detected in cultured mouse PASMCs in the absence and presence of store-depletion. Right: bar graph showing expression levels of STIM1 measured relative to GAPDH in control cells (denoted as 1, filled bar) and in cells subjected to store-depletion (open bar). Data are means ± SE of 7 separate Western blot analyses. C: STIM1 coimmunoprecipitated Orai1 in cultured mouse PASMCs in the absence and presence of store-depletion. STIM1 was first immunoprecipitated (IP) with EXBIO STIM1 antibody (10 μg), and the blot was subsequently probed with BD Biosciences STIM1 antibody (WB, 1:100). The blot was then probed for coimmunoprecipitation (co-IP) of Orai1 expression using Orai1 antibody (WB, 1:100, ProSci). Experiments were performed in 3 separate co-IP procedures and Western blot analyses.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Cell Culture, Expressing, Control, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: American Journal of Physiology - Cell Physiology

Article Title: Orai1 interacts with STIM1 and mediates capacitative Ca 2+ entry in mouse pulmonary arterial smooth muscle cells

doi: 10.1152/ajpcell.00548.2009

Figure Lengend Snippet: Colocalization of Orai1 and STIM1 in mouse PASMCs. A–D: staining of mouse cultured PASMCs after exposure of live cells with normal bath physiological salt solution (PSS). A: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. B–D: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Orai1 and STIM1 colocalization (yellow/orange) is shown in the merged images. E–H: staining of mouse cultured PASMCs after exposure of live cells with Ca2+-free PSS containing 10 μM CPA. E: omission of Orai1 and STIM1 antibody resulted in no Orai1 or STIM1 staining. F–H: three representative cells dual-labeled with anti-Orai1 antibody (green) and STIM1 antibody (red). Colocalization of Orai1 and STIM1 is more apparent (yellow/orange) after store-depletion as shown in the merged images. Nuclei were stained with DAPI (blue). Experiments were performed in 3 separate immunostaining procedure, each with duplicate coverslips. Scale bars, 20 μm.

Article Snippet: To demonstrate coimmunoprecipitation of STIM1 and Orai1, the blot was subsequently probed with Orai1 antibody (1:100, ProSci).

Techniques: Staining, Cell Culture, Labeling, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

doi: 10.3390/ijms21093315

Figure Lengend Snippet: MDA-MB-231 cells express the three components of the arachidonate-regulated Ca 2+ -selective (ARC) channels. MCF10A, MCF7, and MDA-MB-231 cells were seeded in 6-well plates and, upon reaching the adequate cell confluence (90%), they were detached, lysed with NP-40, and denaturated by mixing with Laemmli´s buffer (LB). Subsequent Western blotting (WB) was performed using the anti-STIM1, anti-Orai1, and anti-Orai3 antibodies as described in the Materials and Methods Section. Membranes were reprobed with an anti-β-actin antibody that was used as the loading protein control. Images are representative of 4–6 independent experiments and the histogram represents the fold increase of the protein concentration found with respect to the MCF10A cells. ** p < 0.01, *** p < 0.001 using ANOVA and Tukey’s post-test, respectively.

Article Snippet: Mouse monoclonal anti-STIM1 antibody (catalogue number 6140954, epitope: amino acid 25-139) was purchased from BD-Bioscience ® (Madrid, Spain).

Techniques: Western Blot, Protein Concentration

Journal: International Journal of Molecular Sciences

Article Title: Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

doi: 10.3390/ijms21093315

Figure Lengend Snippet: MDA-MB-231 cells artificially expressing ARC channels present AA-evoked changes in the [Ca 2+ ] c . MDA-MB-231 cells were transfected with either the empty vectors (Mock), the overexpression plasmid of GECO-Orai3, or with the triad of overexpression plasmid for reconstituting the ARC channels (Cherry-STIM1, Orai1-CFP, and GECO-Orai3; GECO-ARC). Upon confirming positive expression of the plasmids using epifluorescence microscopy, cells were maintained in a medium containing 50 µM of CaCl 2 . Cells were alternatively excited at 488 nm (GECO-Orai3 dye; A and C ) and 340/380 nm (Fura-2; B and C ) and fluorescence emitted by the samples was acquired at 505 nm for both fluorescent dyes. At the beginning of the experiments, extracellular medium was supplemented with EGTA (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; 100 µM) and samples were subsequently incubated for 3 min with AA (8 µM). Next, we added 1 mM of CaCl 2 to the extracellular medium and we monitored the Ca 2+ entry evoked by AA stimulation for the next 2 min. Traces are representative of three independent experiments where 2–8 positive transfected cells in each field were analyzed. Bars represent 30 µm.

Article Snippet: Mouse monoclonal anti-STIM1 antibody (catalogue number 6140954, epitope: amino acid 25-139) was purchased from BD-Bioscience ® (Madrid, Spain).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Epifluorescence Microscopy, Fluorescence, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Arachidonic Acid Attenuates Cell Proliferation, Migration and Viability by a Mechanism Independent on Calcium Entry

doi: 10.3390/ijms21093315

Figure Lengend Snippet: Combination of AA and siOrai3 reduces MDA-MB-231 cell proliferation. ( A ) MDA-MB-231 cells were transfected with the siRNA A (control) or siOrai3 for 48 h ( A.1 ) or 96 h ( A.2 ), respectively. WB using the anti-Orai3 antibody was done as described in the Materials and Methods, and following, reprobing of the membranes with an β-actin-antibody was conducted for protein loading control. ( B ) MDA-MB-231 cells were transfected with the siRNA A (control and AA) or siOrai3 for 48 h. Then, an equal number of cells were shed in a 96-well plate and were allowed to proliferate in the absence or presence of AA (8 μM). At the indicated time points (0, 24, and 48 h), cells were incubated with BrdU for 2 h. The histogram represents the mean ± S.E.M. (standard error of the mean) of BrdU uptake of eight independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 respect the BrdU values found in the control at time 0. $$$ p < 0.001 with respect to the BrdU values found in the control at each given time point. ∞ p < 0.05 with respect to the BrdU values found in cells non-genetically modified but treated with AA. We used here the ANOVA and Dunnett’s post-test. ( C ) MDA-MB-231 cells were cultured for 48 h in the presence or absence of AA (8 μM), and cells were subsequently lysed. Upon protein normalization, WB using the anti-STIM1, anti-Orai1, and anti-Orai3 antibodies were performed as described in the Materials and Methods. Bar graph represents the fold increase ± S.E.M. of the protein expression with respect to the control cells non-treated with AA, which did not report statistical significance upon analyzing with ANOVA and Tukey’s post-test.

Article Snippet: Mouse monoclonal anti-STIM1 antibody (catalogue number 6140954, epitope: amino acid 25-139) was purchased from BD-Bioscience ® (Madrid, Spain).

Techniques: Transfection, Incubation, Genetically Modified, Cell Culture, Expressing

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A-B . Confocal images of Immunostaining of endogenous Sec22b using the Synaptic Systems (SYSY) antibody ( A , magenta) or fluorescence of mCherry (mCh)-Sec22b ( B , magenta) in phagocytic MEFs overexpressing GFP-ERGIC-53 (green) and exposed to IgG-beads for 30 min. Sec22b periphagosomal accumulation containing (arrow 1) and devoid (arrow 2 in A, and arrow in B) of the ERGIC marker. Slice 5 of the total stack is shown to highlight total cell staining while Slice 7 is shown in the inset to highlight periphagosomal accumulation. C ) Immunostaining of endogenous Sec22b (green) in phagocytic MEFs exposed to IgG-beads for 30 min with the Santa Cruz antibody (SC). D ) Immunostaining of endogenous Sec22b (white) in phagocytic MEFs overexpressing both the ER-marker RFP-KDEL (magenta) and GFP-ERGIC-53 (green), exposed to IgG-beads for 30 min. Arrow: periphagosomal accumulations of ER marker that are devoid of ERGIC marker and contain a faint but visible Sec22b signal. E ) MEFs overexpressing FCGR2A-GFP (FcR, green) and mCh-Sec22b (magenta) and exposed to IgG-beads for 15 min. F) 3D-CLEM analysis of phagocytic Stim1 -/- ; Stim2 -/- (DKO) MEFs overexpressing mCh-Sec22b (magenta) and YFP-STIM1 (green) exposed to IgG-beads for 30 min. Arrow 1: periphagosomal accumulation of Sec22b co-localizing with STIM1 in an MCS structure. Arrow 2: MCS containing only STIM1 but not Sec22b. One deconvolved confocal slice corresponds approximately to 75 EM slices. Slice 261 of the EM stack is shown in the large images and insets of site 1, while slice 236 is shown for site 2. Insets labelled “a” show an overlay of the EM with the green channel only, those labelled “b” with the red channel only. For A-E bars = 3 µm, for F bars = 1 µm.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Immunostaining, Fluorescence, Marker, Staining

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A) Representative Western blots of SKO ( Stim1 -/- ) and DKO ( Stim1 -/- ; Stim2 -/- ) MEF cell lines stably expressing shCTR and shSec22b incubated with anti-Sec22b (SYSY) and anti-tubulin as control show efficient Sec22b knockdown (quantification in ). B) Representative Western blots of WT, SKO and DKO stable cell lines incubated as above and with anti-STIM1 (left panel) or anti-STIM1 and STIM2 (right panel) confirm the genotype of the cells and show expression of STIM1 is not upregulated upon Sec22b knockdown (quantification in ). C) Related to , quantification of the number (left panel) and kinetics (right panel, t 50% parameter of the Boltzman sigmoidal curve fit) of STIM1 puncta appearing at the TIRF plane after addition of Tg. In WT cells overexpression of Sec22b increases the number of STIM1 puncta, whereas in DKO cells the number is similar but the kinetics are faster. D) Quantification of the kinetics (slope and t 50% parameters of Boltzmann sigmoidal curve fits) of STIM1 puncta arrival at the TIRF plane in response to Tg shows Sec22b knockdown does not impact STIM1 recruitment. Related to . Bar graphs show means +SEM, * p<0.05, **p<0.01.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Western Blot, Stable Transfection, Expressing, Incubation, Over Expression

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A-C) TIRF analysis of wild-type (WT) and DKO MEFs transfected with YFP-STIM1 in combination with ER marker RFP-KDEL or mCh-Sec22b. Cells washed in calcium-free imaging buffer (CF) were imaged every 15 sec, and 1 µM thapsigargin (Tg) was added after 1 min. The number ( B, See also ), size ( C, left panel), and kinetics ( C, right panel) of STIM1 puncta appearance at the TIRF plane, representing ER-plasma membrane (PM) MCS, were quantified 6 min after Tg addition. Larger STIM1 puncta appeared more rapidly upon Sec22b overexpression. In ( A ) error bars are omitted for clarity, mean values and Boltzman sigmoidal curve used to calculate slope parameters are shown. n=8/11;3/5 coverslips WT;DKO KDEL/Sec22b). D) Similar experiments as in A-C revealed Sec22b knockdown in DKO cells does not impact mCh-STIM1 recruitment to ER-PM MCS (n=13/12 shCTR/shSec22b, see also ). E-F) Global store-operated calcium influx was measured using classic Fura-2-based Tg-Calcium re-addition assay (see methods) in WT MEFs transfected with YFP-STIM1 and RFP-KDEL or mCh-Sec22b ( E ) or in WT MEFs transfected only with GFP-KDEL, EGFP-Sec22b, or EGFP-Sec22b-P33 MCS-disrupting mutant ( F ). Influx was faster compared to KDEL and higher compared to wild-type Sec22b in cells expressing the P33 mutant. Error bars are omitted from traces for clarity. G ) Similar measurements of global calcium influx were performed in WT and Stim1 -/- (SKO) cells. Peak influx was higher in WT shSec22b cells (n=15/13;19/20 WT;DKO shCTR/Sec22b). H ) Quantification of Western blots of WT MEF shCTR and shSec22b lysates show no difference in STIM1 expression upon Sec22b knockdown (n=8, blots normalized to anti-tubulin controls, expressed as relative to shCTR, See also ). I ) Quantification of Western blots of SKO and DKO MEF shCTR and shSec22b lysates show robust knockdown (n=5/5 SKO/DKO, blots normalized to anti-tubulin controls, expressed relative to shCTR, see also ). Bar graphs show means +SEM, *p<0.05, *** p<0.001.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Transfection, Marker, Imaging, Over Expression, Mutagenesis, Expressing, Western Blot

Journal: bioRxiv

Article Title: The SNARE Sec22b regulates phagosome maturation by promoting ORP8-mediated PI(4)P exchange at ER-phagosome contact sites

doi: 10.1101/2022.04.12.487993

Figure Lengend Snippet: A) Representative Western blots of WT MEFs transfected with siCTR or siSec22b (50 nM) for 48 h incubated with anti-Sec22b (SYSY) and anti-tubulin as control (related to and ). B-C) Representative Western blots of WT MEFs transfected with siCTR (100 nM) or siORP5/8 (50+50 nM) for 48 h incubated with anti-ORP8 (B) or and anti-ORP5 (C) and anti-tubulin as control (related to ). In (B) the two bands correspond roughly to the molecular weight of the canonical (100 kDa, closed arrow) and short (80 kDa) isoform (ORP8s, open arrow). In (C) the band corresponding to the typical running molecular weight of the canonical ORP5 isoform (110 kDa, closed arrow) responded to siRNA treatment while two other bands (presumed non-specific) did not. D) Quantification of Western blots sets represented in A-C shows efficient knockdown for Sec22b and canonical ORP isoforms (n=8;7;4;3 for siCTR;siSec22b;ORP8 in siORP5/8; ORP5 in siORP5/8). E) Confocal images of mCh-STIM1 and shR-EGFP-Sec22b-P33 in shSec22b MEFs exposed to IgG-beads for 30 min (related to ). Arrows: periphagosomal puncta reminiscent of MCS show co-localization of STIM1 and P33. White bar = 3 µm. Bar graphs are means + SEM. *p<0.05, **p<0.01.

Article Snippet: The following antibodies (antibody name/catalog#/dilution) were purchased from: Synaptic Systems (SYSY): rabbit anti-Sec22b (186003/1:1000); Santa Cruz: mouse anti-Sec22b (29-F7) (sc-101267/1:100); Cell Signaling: mouse anti-c-myc antibody (9B11) (2276/1:100), rabbit anti-STIM2 (4917S/1:1000); Thermo: mouse anti-CD16-CD32 (Fc-Block, 14-0161-85, 1:200), rabbit anti-ORP5 (PA5-18221/1:500), mouse-anti-MHC Class I-PE (H-2Kb, AF6-88.5.5.3, 12-5958-80, 1:100) goat-anti-rabbit Alexa Fluor 555 (A21428/1:1000), goat-anti-mouse Alexa Fluor 647 (A21235/1:1000); GeneTex: rabbit anti-ORP8 (GTX121273/1:500); Sigma: mouse anti-α-tubulin (T9026/1:5000), rabbit anti-sheep red blood cell (sRBC, S1389/1:40), mouse anti-FLAG-M2 (F1804/1:1000); mouse anti-GFP (11814460001/1:1000); BD Biosciences: mouse anti-GOK/STIM1 (610954/1:100); Bio-Rad: goat anti-rabbit IgG (H+L) HRP conjugate (170-6515/1:10000), goat anti-mouse IgG (H+L) HRP conjugate (170-6516/1:10000), Innovative Research: human IgG protein A purified (hIgG) (IR-HU-GF); Jackson ImmunoResearch: donkey-anti-mouse Alexa Fluor 488 (715-545-150/1:800), goat-anti-rabbit Alexa Fluor 647 (111-605-003/1:1000).

Techniques: Western Blot, Transfection, Incubation, Molecular Weight