dync1i1 polyclonal antibody  (Proteintech)

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: Antibody reagents

Article Snippet: Hs00189392_m1 , DYNC1I1.

Techniques: Western Blot, Immunohistochemistry-IF

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: TaqMan assays

Article Snippet: Hs00189392_m1 , DYNC1I1.

Techniques:

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: Sequences of primers used in qPCR

Article Snippet: Hs00189392_m1 , DYNC1I1.

Techniques: Sequencing

Journal: British Journal of Cancer

Article Title: Altered expression of vesicular trafficking machinery in prostate cancer affects lysosomal dynamics and provides insight into the underlying biology and disease progression

doi: 10.1038/s41416-024-02829-x

Figure Lengend Snippet: SiRNA reagents

Article Snippet: Hs00189392_m1 , DYNC1I1.

Techniques:

Journal: The EMBO Journal

Article Title: Annexin A7 enhances TIA1 axonal trafficking to counteract pathological aggregation in neurons

doi: 10.1038/s44318-025-00609-8

Figure Lengend Snippet: ( A ) Venn diagram showing four shared interactors between TIA1 and DIC1B interactomes (DYNC1i1 in BioGRID, see Methods and Dataset ). ( B ) Mass spectrometry analysis of proteins interacting with GST-TIA1 in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and log 2 fold change >1.2). Data from three replicates; statistical significance assessed by paired t -test. ( C ) Immunoblots of ANXA7 in proteins pulled down by GST-TIA1 from rat brain. ( D ) Mass spectrometry analysis of proteins interacting with GST-ANXA7 in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and log 2 fold change >1.2). Data from three replicates; statistical significance assessed by paired t -test. ( E ) Immunoblots of TIA1 and DIC1B in proteins pulled down by GST-ANXA7 from rat brain. ( F ) Key frames from time-lapse images showing retrograde co-trafficking of light-induced Opto-TIA1 (red) and ANXA7-EGFP (green) granules in DIV9 rat hippocampal neurons. Scale bar = 2 µm. Arrowheads indicate co-trafficking. ( G ) Purified recombinant Myc-ANXA7 (rMyc-ANXA7) protein enhances rTIA1 and rFlag-DIC1B interaction, shown by increased rTIA1 pulled down by rFlag-DIC1B. The arrow indicates the weak interaction between TIA1 and DIC1B observed in the absence of rANXA7. ( G’ ) Quantification of ( G ), data from three biological replicates (Myc-ANXA7 vs. TIA1: P = 0.0088; TIA1 vs. TIA1 + Myc-ANXA7: P = 0.0463). ( H ) Co-IP assay examining the interaction between endogenous DIC1B and HA-tagged TIA1 using anti-HA magnetic beads in DIV11 rat cortical neurons. The interaction is studied under endogenous ANXA7 knockdown (shANXA7) or Myc-ANXA7 overexpression conditions. ( H ’) Quantifying TIA1-DIC1B interaction from ( H ) shows the effects of different ANXA7 levels ( n = 5 technical replicates from four biological replicates. Control vs. Myc-ANXA7: P = 0.0267; Control vs. shANXA7: P = 0.0474). ( I ) Schematic diagram of FLIM-FRET to examine the affinity between EGFP-TIA1 (donor) and DIC1B-mRFP (acceptor) under varying levels of ANXA7 (A7). ( J ) Represented images showing color-coded EGFP-TIA1 lifetime in axon shafts of transfected neurons, with lifetime ( J’ ) and FRET efficiency ( J” ) quantified and compared across indicated groups. Scale bar = 2 μm ( n = 29, 37, 35, and 49 axons from four biological replicates. (1) vs. (2): P = 0.0266; (1) vs. (4): P < 0.0001). ( K ) Left: schematic illustrating PLA detection of endogenous TIA1 and DIC1B interaction. Right: representative confocal images showing TIA1/DIC1B PLA signals in neurons with varying ANXA7 (A7) levels; bracketed axons are enlarged below. Scale bars = 50 µm (top), 10 µm (bottom). ( K’ ) Quantification of axonal PLA density from ( K ) ( n = 172, 90, 172, and 90 ROIs from six biological replicates. All P < 0.0001). Data represent mean ± SEM; one-sample t -test in ( G’ , H’ ); one-way ANOVA in ( J’ , J” , K’ ). .

Article Snippet: DYNC1I1 Polyclonal antibody , Proteintech , Cat#13808-1-AP; RRID: AB_2093492.

Techniques: Mass Spectrometry, Control, Western Blot, Purification, Recombinant, Co-Immunoprecipitation Assay, Magnetic Beads, Knockdown, Over Expression, Transfection

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) HDAC6- or DYNC1I1-depleted A549 cells were infected with IAV at a multiplicity of infection (MOI) of 0.1 for 4 hours (h). NS1 and NP mRNA levels were quantified by RT-PCR, normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and expressed relative to siCtrl cells. ( B ) Cells in (A) were infected for 8 hours before WB with anti HDAC6, DYNC1I1, NP, and β-actin antibodies. NP levels were quantified relative to siCtrl cells. ( C ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf, 1 μM Noc, or 10 μM CilioD before WB analysis using anti-NP and anti-GAPDH antibodies. ( D ) NP levels in (C), relative to untreated cells (Ctrl). ( E ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 10 μM CilioD, 1 μM Noc, 0.1 μM LatA, or 1 μM Noc and 0.1 μM LatA simultaneously before WB analysis with anti-NP and anti-GAPDH antibodies. ( F ) NP levels in (E), relative to untreated cells (Ctrl). ( G ) The cytoplasm entry assay was carried out in siCtrl-, siHDAC6-, or siDYNC1I1-treated sHeLa cells, which were then processed for IF with anti-M1 and anti-LAMP1 antibodies. Baf (200 nM) was used under the siCtrl condition to block IAV cytoplasm entry. Insets highlight M1-positive LAMP1 puncta. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( H to J ) Quantification of the cell percentage with dispersed M1 (H), the number of M1 puncta per cell (I), and the percentage of M1-positive LAMP1 puncta (J) in (G). Error bars represent SDs [ n = 3 in (A), (B), (D), and (F); n = 10 in (H) to (J), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Blocking Assay, Microscopy

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa cells treated as in in the presence or absence of Baf were processed for IF with anti-M1 and anti-LC3 antibodies. Insets highlight the colocalization between LC3 and M1, and white arrowheads point to colocalization (in untreated cells) and noncolocalization (in Baf-treated cells). Images were collected using a DeltaVision microscope. Scale bars, 5 μm. ( B ) Quantification of the LC3-positive M1 puncta in (A). Error bars represent SDs. ( C ) IAV cytoplasm entry in sHeLa cells was carried out as in , except that IAV was at an MOI of 30. Baf (200 nM) was used to block IAV fusion at LEs. Cell extracts were subjected to IP with LC3 antibodies before separating the coisolated proteins and WB analysis with anti-LC3, NP, and immunoglobulin G (ΙgG) (control) antibodies. ( D ) Quantification of the NP bound to LC3 in (C), expressed relative to infected cells not treated with Baf. ( E ) Cell extracts from sHeLa cells treated and processed as in (C). WB membranes were probed with anti-LC3, HDAC6, DYNC1I1, KIF5B, and ΙgG antibodies (control). ( F ) Quantification of the DYNC1I1 bound to LC3s in (E), expressed relative to mock-treated cells. Error bars represent SDs [ n = 3 in (D) and (F); n = 3 in (B), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Microscopy, Blocking Assay, Control, Infection

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) Working flow that led to the identification of PCNT as a factor in IAV cytoplasm entry. ( B ) DYNC1I1-, CDK1-, PCNT-, or PPP1CC-depleted sHeLa cells were infected with IAV at an MOI of 10 and processed for IF with the anti-M1 antibody at 3 hpi as in . Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( C and D ) Quantification of both the percentage of cells with dispersed M1 (C) and the amount of M1 puncta per cell (D) in (B). ( E ) sHeLa cells were transfected with siCtrl, siPCNT, or siDYNC1I1 for 48 hours, infected, and processed for ExM as in . Baf (200 nM) was used to block IAV fusion at LEs in sHeLa cells. Scale bars, ~4.5 μm (maximum projection images) and ~0.2 μm (inset images). ( F ) Quantification of the distance of luminal M1 puncta from the LAMP1-positive LE membrane in single slices of expanded cells shown in (E). ( G ) DYNC1I1- or PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 8 hours. Cell extracts were examined by WB with anti-NP, M2, PCNT, DYNC1 I1, and β-actin antibodies. ( H ) NP and M2 level quantification in (G). Bars represent average amounts relative to infected cells treated with siCtrl. ( I ) PCNT-depleted sHeLa cells were infected with luc-HSV-1 or luc-VaV at an MOI of 1 for 6 hours. Luciferase activity in cell extracts was then measured. Data represent the average luciferase activities expressed relative to the siCtrl for each virus. Error bars represent SDs [ n = 10 in (C) and (D), 50 cells counted per repeat; n = 3 in (F), (H), and (I)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Microscopy, Transfection, Blocking Assay, Membrane, Luciferase, Activity Assay, Virus

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) sHeLa and A549 cells were infected with IAV at an MOI of 0.1 for 8 hours in the presence of 200 nM Baf or 100 nM Ctn before WB with the indicated antibodies. ( B ) NP levels in (A) relative to siCtrl. ( C ) PCNT-depleted sHeLa cells transfected with the GFP-PCNTB or GFP-PCNTS plasmid were infected with IAV at an MOI of 0.1 for 8 hours and examined by WB with the indicated antibodies. NP levels are relative to siCtrl. The GFP antibody was used for GFP-PCNTS. The PCNT antibody only detects PCNTB. ( D ) Cells as in (C) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to infected siCtrl cells. ( E ) PCNT-depleted sHeLa cells were transfected with the GFP-PCNTS or GFP-PCNTS ΔPACT plasmid before IAV infection at an MOI of 0.1 for 8 hours and examined by WB analysis with the indicated antibodies. NP levels are relative to siCtrl. ( F ) Cells as in (E) were infected with WSN-luc IAV for 16 hours, and luciferase activity was measured relative to siCtrl. ( G ) DYNC1I1-, PCNT-, or LC3/PCNT-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified by RT-PCR, normalized to GAPDH, and expressed relative to siCtrl. ( H ) Cells from (G) were infected with IAV for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. ( I ) DYNC1I1-, PCNT-, HDAC6-, or PCNT/HDAC6-depleted sHeLa cells were infected with IAV at an MOI of 0.1 for 4 hours, and NP and NS1 mRNA levels were quantified as in (G). ( J ) Cells as in (I) were infected for 8 hours before WB with the indicated antibodies. NP levels are relative to siCtrl. Error bars represent the SDs [ n = 3 in (B) to (J)]. Asterisks indicate significant differences. h, hours.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: Influenza A virus subverts the LC3-pericentrin dynein adaptor complex for host cytoplasm entry

doi: 10.1126/sciadv.adu7602

Figure Lengend Snippet: ( A ) PCNT-depleted atg7 −/− cells were infected with IAV at MOI 30 for 3 hours, and cell extracts were subjected to IP with an anti-LC3 antibody before examining the input and the coisolated proteins by WB with anti-LC3, NP, PCNT, DYNC1I1, and ΙgG (control) antibodies. ( B ) DYNC1I1, NP, and PCNT bound to LC3s in (A) relative to the infected siCtrl cells. ( C ) sHeLa APEX2KI and LC3 APEX2KI cells were infected with IAV as in , but 500 μM biotin phenol (BP) and 1 mM H 2 O 2 were added 30 and 1 min, respectively, before isolating biotinylated proteins. sHeLa APEX2KI cells without BP incubation were used as a negative control. The input and the affinity-purified proteins were analyzed by WB with antibodies against biotin, NP, PCNT, DYNC1I1, or β-actin. ( D and E ) Biotinylated DYNC1I1 (D) and NP (E) in (C) relative to the noninfected sHeLa APEX2KI cells. ( F ) PCNT-depleted atg7 −/− cells were processed for IF as in with antibodies against M1 and LC3. Insets highlight colocalization between M1 and LC3. Images were acquired using a ZEISS LSM800 microscope. Scale bars, 5 μm. ( G ) Percentage of the LC3-positive M1 puncta in (F). ( H ) Model for IAV host cytoplasm entry. The lower pH of LEs triggers the fusion between endocytoses IAV VPs at LEs. Uncoating and cytoplasmic vRNP release is mediated by two dynein-dependent systems that take advantage of the pulling force of MT-based motors. vRNPs are linked to dynein motors via the LC3-PCNT adaptor complex or HDAC6, which binds ubiquitin. It is unknown which vRNP components interact with LC3s and HDAC6. Error bars represent SDs [ n = 3 in (B), (D), and (E); n = 5 in (C), 50 cells counted per repeat]. Asterisks indicate significant differences.

Article Snippet: Proteins of interest were detected using specific antibodies against LC3, GABARAP, NP, M2, ATG7 (Cell Signaling Technology, Danvers, MA, #2631S), ATG13 (Rockland Immunochemicals, Pottstown, PA, #SAB4200100), β-actin (Merck Millipore, #MAB1501), PCNT (Sigma-Aldrich, #HPA016820), GFP (monoclonal; Takara, Shiga, Japan, #632381), GFP (polyclonal; Abcam, #ab6556), DYNC1I1 (Novus, St. Charles, MO, #NBP1-87972), HDAC6 (Abcam, #ab1440), biotin (Rockland, #100-4198), vinculin (Cell Signaling Technology, #13901S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Thermo Fisher Scientific, #4333764T), and secondary antibodies conjugated to Alexa Fluor 680 or Alexa Fluor 800 (Molecular probes).

Techniques: Infection, Control, Incubation, Negative Control, Affinity Purification, Microscopy, Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Axon Trafficking Counteracts Aberrant Protein Aggregation in Neurons

doi: 10.1101/2025.01.16.633295

Figure Lengend Snippet: (A) Venn diagram showing four shared interactors between TIA1 and DIC1B interactomes (DYNC1i1 in BioGrid). (B, D) Mass spectrometry analysis of proteins interacting with GST-TIA1 (B) or GST-ANXA7 (D) in rat brain lysates, using GST tag as a control. Red dots indicate significantly enhanced interactors ( p < 0.05 and Log2 Fold Change > 1.2). Data from three replicates. (C, E) Immunoblots of ANXA7 in proteins pulled down by GST-TIA1 (C) ; TIA1 and DIC1B in proteins pulled down by GST-ANXA7 (E) from rat brain. (F) GO and KEGG pathway analysis of GST-ANXA7 interactors, including Biological Processes (BP), Cellular Components (CC), and Molecular Functions (MF). (G) Co-IP showing Myc-ANXA7 and HA-DIC1B interact with Flag-TIA1 in HEK293T cells. (H) Confocal images of endogenous TIA1 (green) and ANXA7 or DIC1B (red) in the cortex and hippocampus of P34 mouse brain. Scale bars = 500 μm and 10 μm. (I) Time-lapse images showing retrograde co-trafficking of light-induced Opto-TIA1 (red) and ANXA7-GFP (green) granules in DIV9 rat hippocampal neurons. Scale bar = 2 µm. Arrowheads indicate co-trafficking. (J) In vitro protein pull-down assay schematic. (K) Purified Myc-ANXA7 protein enhances TIA1 and Flag-DIC interaction, shown by increased TIA1 co-IP’d with Flag-DIC. (L) Co-IP assay examining the interaction between endogenous DIC1B and HA-tagged TIA1 using anti-HA magnetic beads in cultured DIV11 rat cortical neurons. The interaction is studied under endogenous ANXA7 knockdown (shANXA7) or Myc-ANXA7 overexpression conditions. (M) Quantifying TIA1-DIC1B interaction from (L) shows the effects of different ANXA7 levels (n = 5, 4, 4). (N) Schematic diagram of FLIM-FRET to examine the affinity between GFP-TIA1 (donor) and DIC1B-mRFP (acceptor) under varying levels of ANXA7. (O) Represented images showing colour-coded GFP-TIA1 lifetime in axon shafts of transfected neurons, with lifetime (P) and FRET efficiency (Q) quantified and compared across indicated groups. Scale bars =2 μm (n = 29, 37, 35, 49). Data represent mean ± SEM; in (M) two-tailed unpaired t -test; in (P, Q) one-way ANOVA. * p <0.05, *** p <0.001, ns non-significant.

Article Snippet: Primary antibodies: anti-TIA1 (#sc-166247, Santa Cruz), anti-TIA1 (#12133-2-AP, Proteintech), anti-SQSTM1/p62 (#A11247, ABclonal), anti-G3BP1 (#sc-365338, Santa Cruz), anti-Rab5 (#3547, Cell Signaling Technology), anti-LC3B (#83506, Cell Signaling Technology), anti-LAMP1 (#ab13523, Abcam), anti-DYNC1I1 (#13808-1-AP, Proteintech), anti-ANXA7 (#10154-2-AP, Proteintech), anti-GAPDH (#10494-1-AP, Proteintech), anti-HA (#3724, Cell Signaling Technology), anti-Myc (#60003-2-Ig, Proteintech), anti-Myc (#16286-1-AP, Proteintech), anti-Flag (#20543-1-AP, Proteintech), anti-TDP-43 (#89789, Cell Signaling Technology), anti-Iba1 (#019-19741, FUJIFILM).

Techniques: Mass Spectrometry, Control, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Pull Down Assay, Purification, Magnetic Beads, Cell Culture, Knockdown, Over Expression, Transfection, Two Tailed Test

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 3. PGC-1α increases the levels of mitochondrial transport proteins in the cortex of APP/PS1 mice. Immunohistochemistry was used to examine the expression patterns and levels of (Aa-a’) MFN2, (Ba-a’) KIF5A, and (Ca-a’) Dync1i1 in cortex samples from WT/2 × Tg-AD mice. The impact of treatment (AAV-Vector/AAV-PGC-1α) on the expression and levels of (Ab-b’) MFN2, (Bb- b’) KIF5A, and (Cb-b’) Dync1i1 in cortex samples from 2 × Tg-AD mice was also assessed using immunohistochemistry. Scale bars = 100 μm. The expression patterns and qualification of (Bc-c’) KIF5A in cortical samples from 2 × Tg-AD mice treated with AAV-Vector/AAV-PGC-1α were examined with immunoflu orescence. Scale bars = 200 μm. Green = HA-labeled PGC-1α; Red = KIF5A; Blue = DAPI. (Bd-d’) N2A cells were transfected with pEnCMV/PGC-1α plasmid and plasmid-encoding APPswe for 48 h. The expression patterns and quantifi cation of KIF5A in the cells were studied with western blot. For each group, n = 6. Values are expressed as the means ± S.E.M. Significance levels were set at ** p < 0.01 and *** p < 0.001 for noted differences between AAV-Vector- and AAV-PGC-1α-infused AD mice or pEnCMV- or PGC-1α-transfected APPswe cells. GAPDH was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Labeling, Transfection, Western Blot, Control

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 5. RID3, but not RID2, of the PGC-1α region is indispensable for promoting retrograde transport of axonal mitochondria and enhancing mitochondrial auto phagic clearance. N2A cells were co-transfected with plasmid encoding APPswe and pEnCMV/PGC-1α/PGC-1αmL2/PGC-1αmL3 plasmids. The lysates were subjected to immunoblotting using the specified antibodies. Expression patterns and quantification of (A) the retrograde transport protein Dync1i1 and (B) mitophagy-relevant proteins, including (a) Parkin, (b) Pink, (c) LC3-I/II, (d) Beclin and (e) P62, were examined via western blot analysis. GAPDH was utilized as a loading control. Each group consisted of n = 6 samples. (C) Fluorescence confocal microscopy was employed to detect JC-1 signals in N2A cells. Data represent three independent measurements. Fluorescence was captured using excitation at 488 nm and adjusting the emission of confocal microscopy for J-monomers (visible as green) and J- aggregates (visible as red/orange). (D) The ratios of J-aggregates to J-monomers were quantified as an indicator of mitochondrial membrane potential (MMP). Each group included n = 6 samples. Scale bars = 10 μm. The data underwent One-way ANOVA analysis to assess the effects of individual factors in isolation from others. Different letters indicate significant differences in mean values between groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Fluorescence, Confocal Microscopy, Membrane, Isolation

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 3. PGC-1α increases the levels of mitochondrial transport proteins in the cortex of APP/PS1 mice. Immunohistochemistry was used to examine the expression patterns and levels of (Aa-a’) MFN2, (Ba-a’) KIF5A, and (Ca-a’) Dync1i1 in cortex samples from WT/2 × Tg-AD mice. The impact of treatment (AAV-Vector/AAV-PGC-1α) on the expression and levels of (Ab-b’) MFN2, (Bb- b’) KIF5A, and (Cb-b’) Dync1i1 in cortex samples from 2 × Tg-AD mice was also assessed using immunohistochemistry. Scale bars = 100 μm. The expression patterns and qualification of (Bc-c’) KIF5A in cortical samples from 2 × Tg-AD mice treated with AAV-Vector/AAV-PGC-1α were examined with immunoflu orescence. Scale bars = 200 μm. Green = HA-labeled PGC-1α; Red = KIF5A; Blue = DAPI. (Bd-d’) N2A cells were transfected with pEnCMV/PGC-1α plasmid and plasmid-encoding APPswe for 48 h. The expression patterns and quantifi cation of KIF5A in the cells were studied with western blot. For each group, n = 6. Values are expressed as the means ± S.E.M. Significance levels were set at ** p < 0.01 and *** p < 0.001 for noted differences between AAV-Vector- and AAV-PGC-1α-infused AD mice or pEnCMV- or PGC-1α-transfected APPswe cells. GAPDH was used as the loading control. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Immunohistochemistry, Expressing, Plasmid Preparation, Labeling, Transfection, Western Blot, Control

Journal: Experimental gerontology

Article Title: The potential benefits of PGC-1α in treating Alzheimer's disease are dependent on the integrity of the LLKYL L3 motif: Effect of regulating mitochondrial axonal transportation.

doi: 10.1016/j.exger.2024.112514

Figure Lengend Snippet: Fig. 5. RID3, but not RID2, of the PGC-1α region is indispensable for promoting retrograde transport of axonal mitochondria and enhancing mitochondrial auto phagic clearance. N2A cells were co-transfected with plasmid encoding APPswe and pEnCMV/PGC-1α/PGC-1αmL2/PGC-1αmL3 plasmids. The lysates were subjected to immunoblotting using the specified antibodies. Expression patterns and quantification of (A) the retrograde transport protein Dync1i1 and (B) mitophagy-relevant proteins, including (a) Parkin, (b) Pink, (c) LC3-I/II, (d) Beclin and (e) P62, were examined via western blot analysis. GAPDH was utilized as a loading control. Each group consisted of n = 6 samples. (C) Fluorescence confocal microscopy was employed to detect JC-1 signals in N2A cells. Data represent three independent measurements. Fluorescence was captured using excitation at 488 nm and adjusting the emission of confocal microscopy for J-monomers (visible as green) and J- aggregates (visible as red/orange). (D) The ratios of J-aggregates to J-monomers were quantified as an indicator of mitochondrial membrane potential (MMP). Each group included n = 6 samples. Scale bars = 10 μm. The data underwent One-way ANOVA analysis to assess the effects of individual factors in isolation from others. Different letters indicate significant differences in mean values between groups. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: PGC-1a (Bioss, cat # bs-1832R, Beijing, China), Aβ (CST, cat # D3D2N, Boston, USA), HA (Boster, cat # bsm-33,003 M, Beijing, China), BAX (Abcam, cat # ab32503, Cambridge, MA, USA), Bcl-2 (Abcam, cat # ab182858, Cambridge, MA, USA), MFN2 (Proteintech, cat # 12186–1-AP, Rosemont, USA), KIF5A (Bioword, cat # BS71526, Nanjing, China), KIF5B (Wanleibio, cat # WL04906, Shenyang, China), Dync1i1 (Proteintech, cat # 13808–1-AP, Rosemont, USA), Flag (abm, cat # G188, Zhenjiang, China), Tubulin (abm, cat # G098, Zhenjiang, China), GFP (Beyotime, cat # AG281, Shanghai, China), GAPDH (Boster, cat # BM1623, Wuhan, China), Parkin (Wanleibio, cat # WL02512, Shenyang, China), Pink (Wanleibio, cat # WL04963, Shenyang, China), Beclin (ABclonal, cat # A7353, Wuhan China), P62 (ABclonal, cat # A7758, Wuhan China), LC3-I/II (Cell Signaling Technologies Inc., cat # 12741, Beverly, MA, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Control, Fluorescence, Confocal Microscopy, Membrane, Isolation