Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: ALG-2-binding assays of SGFP2-fused proteins containing ABM-2 sequences. Full lengths (-f) or partial fragments (-p) of ALG-2-binding candidate proteins that were fused with SGFP2 or unfused negative control SGFP2 (Ctrl) were transiently expressed in HEK293 ALG-2KD cells. After SGFP2-fused proteins had been immunoprecipitated with anti-GFP pAb from cleared cell lysates as described in Materials and Methods, proteins bound to the protein G-immobilized magnetic beads were subjected to ALG-2-binding assays by ( A ) Far Western blotting (FW) and by ( B ) pulldown assays. ( A ) Blotted membranes were probed with Nluc-ALG-2 for FW (upper panel) and anti-GFP mAb (lower panel) for Western blotting (WB). ( B ) The amounts of Nluc-ALG-2 bound to the beads were quantified by measuring luciferase activities. Binding activities relative to Sec31A-p (100%) were calculated and data were expressed as mean ± SE ( n = 3). Binding capacities compared to the negative control were statistically evaluated: * p < 0.01; ** p < 0.001.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Binding Assay, Negative Control, Immunoprecipitation, Magnetic Beads, Far Western Blot, Western Blot, Luciferase

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Co-IP of ALG-2 and SARAF with anti-SARAF polyclonal antibody. ( A ) Schematic diagram of SARAF showing the region of antigen used for polyclonal antibody (pAb) preparation and mutants lacking the sequence of the ALG-2-binding motif type 2 (ΔABM-2) or amino acid substitution (F228S). SP, signal peptide; TM, transmembrane. ( B , C ) Co-IP assays using HEK293 cells expressing untagged wild type (WT) SARAF or mutants (ΔABM-2, F228S) were performed as described in Materials and Methods. Antibodies (control rabbit IgG , anti-SARAF pAb) were added to the cleared cell lysates ( Input ) that were supplemented with 100 μM CaCl 2 ( Ca ) or 5 mM EGTA ( Eg ), and immunoprecipitated proteins ( IP ) were analyzed by WB with anti-ALG-2 pAb (upper panel) and anti-SARAF pAb (lower panel). IgG-L , IgG light chain. Filled arrowhead, SARAF; unfilled arrow head, ALG-2; asterisks, non-specific. ( D ) HeLa cells and MCF7 cells were used for co-IP assays of endogenously present ALG-2 and SARAF as described in B and C except for no DNA transfection. IgG , control antibody.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Co-Immunoprecipitation Assay, Sequencing, Binding Assay, Expressing, Immunoprecipitation, Transfection

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Reciprocal co-IP assay of ALG-2 and SARAF with anti-ALG-2 antibody. ( A ) HEK293 cells stably expressing untagged SARAF were used for co-IP assays in the presence of 100 μM CaCl 2 ( Ca ) or 5 mM EGTA ( Eg ) with anti-ALG-2 mAb and control mouse IgG. Immunoprecipitates ( IP ) were analyzed by WB with anti-ALG-2 pAb and anti-SARAF pAb as indicated. IgG-L , IgG light chain. ( B ) Lysates of parental HeLa cells and ALG-2 knockout (KO) HeLa cells (ALG-2KO) were subjected to immunoprecipitation with anti-ALG-2 mouse mAb followed by WB with rabbit pAbs against ALG-2, SARAF and Sec31A as indicated. ( C ) A co-IP assay with anti-ALG-2 mAb was performed as shown in ( B ) but with the use of MCF7 cells in the presence of varying concentrations of exogenously added CaCl 2 or 5 mM EGTA as indicated.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Knock-Out, Immunoprecipitation

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Deletion mutation of the SARAF cytosolic domain (CytD) affecting ALG-2 binding and ubiquitination. ( A ) Schematic diagram of the SARAF CytD, which contains three distinct motifs rich in Pro and aromatic residues (designated regions 1, 2 and 3). Expression plasmids for the SARAF CytDs of WT and various deletion mutants that were fused with Strep-tag II and 3xHA (StrepHA) at the N -terminus and SGFP2 at the C-terminus were constructed. ( B ) Strep-pulldown assay. After HEK293 cells had been transfected with pStrepHA-SARAF_CytD-SGFP2 (WT and deletion mutants) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing protease inhibitors, 0.2% Nonidet P-40 and 10 μM CaCl 2 . The cleared cell lysates ( Input ) were subjected to Strep-pulldown followed by WB with anti-ALG-2 and anti-HA as indicated.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Mutagenesis, Binding Assay, Expressing, Strep-tag, Construct, Transfection, Cell Culture, Lysis

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Mutations in the SARAF CytD affecting ubiquitination and suppression of ubiquitination by overexpression of ALG-2. ( A ) Schematic diagram of mutations at ABM-2 (F228S), two PP X Y motifs (Pro-to-Ala mutations) and two potential ubiquitination sites (Lys-to-Arg mutations) in the SARAF CytD. ( B , C ) After HEK293 ALG-2KO cells had been co-transfected with expression plasmids for StrepHA-SARAF_CytD-SGFP (WT or mutants) and with ( B ) FLAG-ALG-2 (WT) or with ( C ) FLAG-ALG-2 mutants and cultured for 24 h, the cleared cell lysates were prepared as described in the legend to B and were subjected to the Strep-pulldown assay followed by WB with anti-HA and anti-FLAG antibodies as indicated. Asterisk, degraded products lacking a Strep tag or non-specific bands; arrowheads, non-ubiquitinated unmodified proteins.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Over Expression, Transfection, Expressing, Cell Culture, Strep-tag

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Evidence of ubiquitination reactions after cell lysis. ( A ) Schematic diagram of HiBiT-Ub assay. ( B ) StrepHA-SARAF_CytD-SGFP2 and HiBiT-tagged ubiquitin (HiBiT-Ub) were individually expressed in HEK293 cells in separate culture dishes. The cleared cell lysates prepared with HKM buffer containing 0.2% Nonidet-P were mixed, and aliquots were subjected to Strep-pulldown in the presence of supplemental chemicals as indicated (5 mM EGTA, 5 mM EDTA, 10 μM CaCl 2 , 10 mM NEM or 3 mM ATP plus 0.2 mM DTT). Pulldown products were resolved by SDS-PAGE followed by Western blotting with respective antibodies as indicated (anti-HA, anti-ALG-2) or by probing with LgBiT for detection of HiBiT-Ub.

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Lysis, SDS Page, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: The Penta-EF-Hand ALG-2 Protein Interacts with the Cytosolic Domain of the SOCE Regulator SARAF and Interferes with Ubiquitination

doi: 10.3390/ijms21176315

Figure Lengend Snippet: Importance of ALG-2-binding to SARAF for Ca 2+ -dependent interaction between the SARAF CytDs. ( A ) Schematic diagram of expressed proteins and co-IP/HiBiT assay. ( B ) After HEK293 cells in 6-cm dishes had been co-transfected with expression plasmids for full-length HiBiTHA-SARAF (WT or F228S mutant) and for either StrepHA-SGFP2 (Control) or StrepHA-SARAF_CytD-SGFP2 (WT or F228S mutant) and cultured for 24 h, the cells were lysed with lysis buffer HKM containing 1% Nonidet P-40 supplemented with protease inhibitors, 10 mM NEM and 10 μM CaCl 2 ( Ca ) or 5 mM EGTA ( Eg ). SGFP2-fused proteins were immunoprecipitated with anti-GFP pAb, and the amounts of co-immunoprecipitated HiBiTHA-SARAF were estimated by the lytic HiBiT assay with LgBiT. The amounts of immunoprecipitated SGFP2-fused proteins were normalized by the StrepTactin-AP method as described in Materials and Methods. The relative amount of HiBiTHA-SARAF WT co-immunoprecipitated with StrepHA-SARAF_CytD-SGFP2 WT was expressed as 100% of relative interaction activity. The co-IP/HiBiT assay was performed in duplicate and repeated three times. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated in three different ways: asterisks for comparison between conditions of plus and minus Ca 2+ ; a for comparison with WT in the presence of Ca 2+ ; b for comparison with the control in the presence of Ca 2+ . p values were below 0.001 for all cases indicated (***, a , and b ). n.s., not significant. ( C ) Enhancement of the Ca 2+ -dependent CytD-to-CytD interaction of SARAF in HEK293 ALG-2KO cells by co-overexpression of FLAG-ALG-2. HEK293 ALG-2KO cells were co-transfected with expression plasmids for (i) full-length HiBiTHA-SARAF (WT), (ii) StrepHA-SARAF_CytD-SGFP2 (WT) and (iii) FLAG empty vector or FLAG-ALG-2 (WT, F85A or Y180A) and cultured for 24 h. The cells were lysed and subjected to HiBiT assay as described above. Relative interaction by co-overexpression of ALG-2 WT in the presence of Ca 2+ was expressed as 100% activity. Data are expressed as mean ± SEM ( n = 3). Statistical significance by Tukey’s test is indicated by asterisks, a , and b as described in ( B ) for comparison with the presence or absence of Ca 2+ (asterisks), WT ( a ) and control ( b ).

Article Snippet: A synthetic gene encoding Nanoluciferase-fused ALG-2 (Nluc-ALG-2, DDBJ/GenBank/EMBL accession No. LC381846) for bacterial expression was synthesized and subcloned into the NcoI/EcoRI site of a T7 RNA polymerase expression vector, pET24d, at GenScript (Piscataway, NJ, USA).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Transfection, Expressing, Mutagenesis, Cell Culture, Lysis, Immunoprecipitation, Activity Assay, Over Expression, Plasmid Preparation