rabbit polyclonal anti ire1α  (Thermo Fisher)

Journal: Frontiers in Physiology

Article Title: Inositol-requiring enzyme 1 alpha is essential for dentinogenesis

doi: 10.3389/fphys.2025.1722417

Figure Lengend Snippet: Immunohistochemistry staining of phosphorylated IRE1α (pIRE1α) protein in the mandibular first molars. (A) Shown are the representative images of immunohistochemistry staining of pIRE1α protein (signal in brown) in the mandibular first molars of 3-week-old Dspp +/+ , Dspp P19L/+ , and Dspp P19L/P19L mice. Each image in (A) is from the middle region of the crown of a sagittally-sectioned mandibular first molar. (A1-A3) are the higher magnification views of the roof-forming odontoblasts (box1), dental pulp cells (box 2) and floor-forming odontoblasts (box 3) in the corresponding images in (A) , respectively. rd, roof dentin; fd, floor dentin; rod, roof-forming odontoblasts; fod, floor-forming odontoblasts; Scale bars: 50 μm in A; 20 μm in (A1-A3). Three independent experiments for IHC staining of pIRE1α show similar results.

Article Snippet: The primary antibodies used in this study included: 1) rabbit anti-phosphorylated IRE1α polyclonal antibody (1:400, Novus Biologicals, NB100-2323); 2) rabbit anti-XBP1 polyclonal antibody that recognizes both unspliced XBP1 (XBP1U) and spliced XBP1 (XBP1S) (1:200, Abcam, ab37152); 3) rabbit anti-XBP1S monoclonal antibody (E9V3E) that specifically recognizes XBP1S ( ; ) (1:50, Cell Signaling Technology, Danvers, MA); 4) rabbit anti-DSPP polyclonal antibody that recognizes both DSP and full-length DSPP (1:2000) ( ); and 5) rabbit anti-DMP1 polyclonal antibody (1:800, #857) ( ).

Techniques: Immunohistochemistry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: Paucity of DNAJC6 upregulates ER α-synuclein or phospho-α-synuclein Ser129 and activates ER stress and the UPR in dopaminergic neurons. ( A ) DNAJC6 deficiency caused by 3-day transfection of DNAJC6 shRNAs significantly increased ER α-synuclein or phospho-α -synuclein Ser129 in dopaminergic neurons. Glucose 6-phosphotase is an ER protein marker. Mitochondrial protein marker COX IV and cytoplasm protein marker β-tubulin were not expressed in the ER. ( B ) Three days after transfection of DNAJC6 shRNA, upregulated protein levels of ER chaperones, including calnexin, Ero1-Lα, Grp78 and PDI, and UPR markers, including IRE1α, PERK, phospho-IRE1α Ser724 and phospho-PERK Thr980 , were observed in dopaminergic neurons. Each bar represents the mean ± S.E. value of 6 experiments. ** p < 0.01 compared with control dopaminergic neurons.

Article Snippet: PVDF membranes were then incubated with the following primary antibodies: (1) rabbit anti-calnexin monoclonal antibody (Cell Signaling Technology); (2) rabbit polyclonal anti-Ero1-Lα antiserum (Cell Signaling Technology); (3) rabbit anti-Grp78 monoclonal antibody (Cell Signaling Technology); (4) rabbit monoclonal anti-PDI antiserum (Cell Signaling Technology); (5) rabbit anti-IRE1α monoclonal antibody (Cell Signaling Technology); (6) rabbit monoclonal anti-PERK antiserum (Cell Signaling Technology); (7) rabbit anti-phospho-IRE1α Ser724 polyclonal antibody (ThermoFisher); (8) rabbit monoclonal anti-phospho-PERK Thr980 antiserum (ThermoFisher); (9) rabbit anti-ATF4 polyclonal antibody (Proteintech, Rosemont, IL, USA); (10) mouse monoclonal anti-CHOP antiserum (Cell Signaling Technology); (11) rabbit anti-caspase-12 polyclonal antibody (Cell Signaling Technology); (12) rabbit monoclonal anti-Bim antiserum (Cell Signaling Technology); (13) rabbit anti-Noxa polyclonal antibody (Invitrogen); (14) rabbit monoclonal anti-Puma antiserum (Cell Signaling Technology).

Techniques: Transfection, Marker, shRNA, Control

Journal: International Journal of Molecular Sciences

Article Title: Downregulation of Protease Cathepsin D and Upregulation of Pathologic α-Synuclein Mediate Paucity of DNAJC6-Induced Degeneration of Dopaminergic Neurons

doi: 10.3390/ijms25126711

Figure Lengend Snippet: PARK19 DNAJC6 mutants fail to inhibit tunicamycin- or rotenone-evoked neurotoxicity, α-synuclein upregulation and apoptotic signaling in dopaminergic neurons. ( A ) FLAG-tagged human WT, Q789X or R927G DNAJC6 was transiently expressed in SH-SY5Y dopaminergic neurons. ( B ) Three-day incubation of 1 μM tunicamycin (TCM) or 0.5 μM rotenone (RTN) induced the neurodegeneration of dopaminergic cells. WT DNAJC6 significantly inhibited tunicamycin- or rotenone-evoked degeneration of dopaminergic neurons. On the contrary, PARK19 DNAJC6 mutants (Q789X or R927G) failed to prevent tunicamycin- or rotenone-triggered neurodegeneration. Each bar shows the mean ± S.E. value of 7 experiments. ( C ) Tunicamycin (TCM; 1 μM) upregulated the cytosolic expressions of active caspase-12, PERK, CHOP, IRE1α, Grp78 and α-synuclein. WT DNAJC6 significantly prevented the tunicamycin-evoked upregulation of cytosolic active caspase-12, PERK, CHOP, IRE1α, Grp78 and α-synuclein. PARK19 DNAJC6 mutants (Q789X or R927G) did not affect the upregulation of cytosolic active caspase-12, PERK, CHOP, IRE1α, Grp78 or α-synuclein caused by tunicamycin. ( D ) Rotenone (RTN; 0.5 μM) upregulated the cytosolic levels of α-synuclein, active caspase-3, active caspase-9 and cytochrome c. WT DNAJC6 inhibited the rotenone-evoked upregulation of cytosolic α-synuclein, active caspase-3, active caspase-9 and cytochrome c. PARK19 DNAJC6 mutants (Q789X or R927G) failed to reverse the upregulation of cytosolic α-synuclein, active caspase-3, active caspase-9 and cytochrome c caused by rotenone. Each bar represents the mean ± S.E. value of 6 experiments. ** p < 0.01 or *** p < 0.001 compared with control dopaminergic neurons. # p < 0.01 compared with tunicamycin- or rotenone-treated dopaminergic neurons.

Article Snippet: PVDF membranes were then incubated with the following primary antibodies: (1) rabbit anti-calnexin monoclonal antibody (Cell Signaling Technology); (2) rabbit polyclonal anti-Ero1-Lα antiserum (Cell Signaling Technology); (3) rabbit anti-Grp78 monoclonal antibody (Cell Signaling Technology); (4) rabbit monoclonal anti-PDI antiserum (Cell Signaling Technology); (5) rabbit anti-IRE1α monoclonal antibody (Cell Signaling Technology); (6) rabbit monoclonal anti-PERK antiserum (Cell Signaling Technology); (7) rabbit anti-phospho-IRE1α Ser724 polyclonal antibody (ThermoFisher); (8) rabbit monoclonal anti-phospho-PERK Thr980 antiserum (ThermoFisher); (9) rabbit anti-ATF4 polyclonal antibody (Proteintech, Rosemont, IL, USA); (10) mouse monoclonal anti-CHOP antiserum (Cell Signaling Technology); (11) rabbit anti-caspase-12 polyclonal antibody (Cell Signaling Technology); (12) rabbit monoclonal anti-Bim antiserum (Cell Signaling Technology); (13) rabbit anti-Noxa polyclonal antibody (Invitrogen); (14) rabbit monoclonal anti-Puma antiserum (Cell Signaling Technology).

Techniques: Incubation, Control

Journal: Frontiers in Physiology

Article Title: Constitutive expression of spliced X-box binding protein 1 inhibits dentin formation in mice

doi: 10.3389/fphys.2023.1319954

Figure Lengend Snippet: IRE1α activation-independent expression of XBP1S. (A, B) . Shown are the schematic representations of the Xbp1 minigene (A) and Xbp1s minigene (B) constructs. The Xbp1s minigene contains a modified exon 4 (Δ26) that lack the 26 intronic sequence, and it also contains a loxP site in intron 3. +1, the transcription start site; E, exon; green boxes, coding exons; and grey boxes, non-coding exons. AscI, NsiI, EcoRV, HindIII, SspI and AflII are restriction endonucleases. (C, D) . HEK 293 cells were transiently transfected with the Xbp1 minigene (C) or Xbp1s minigene (D) together with the pCDNA3 empty vector (Vector) (lane 3) or a construct expressing normal (Norm) IRE1α (lane 4) or two IRE1α variants, K599A (lane 5) and N906A (lane 6). HEK 293 cells were transfected with the pCDNA3 empty vector alone (lane 1) or the construct expressing normal IRE1α alone (lane 2) as controls. Total cell lysate was harvested 48 h after transfection and analyzed by Western-blotting with an antibody that recognizes both XBP1U and XBP1S, the blot was then stripped and sequentially probed with an antibody against phosphorylated IRE1α and total IRE1α. The blot was probed with mouse monoclonal β-actin antibody as the loading control.

Article Snippet: The membrane was sequentially immunoblotted with rabbit anti-XBP1 polyclonal antibody that recognizes both XBP1U and XBP1S (1:4000, Abcam, Cambridge, MA), horseradish peroxidase (HRP)-conjugated rabbit polyclonal anti-phosphorylated IRE1α (pSer724) (1:5000; Novus Biologicals) and mouse monoclonal anti-IRE1α antibody (1:1000; Santa Cruz Biotechnology, Inc.).

Techniques: Activation Assay, Expressing, Construct, Modification, Sequencing, Transfection, Plasmid Preparation, Western Blot, Control