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Proteintech appl1 monoclonal antibody
Appl1 Monoclonal Antibody, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 93 stars, based on 13 article reviews

appl1 monoclonal antibody - by Bioz Stars, 2026-03

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APPL1 transgene expression in the brain. A , Representative Western blot comparing APPL1 expression in various brain regions (CTX, cortex; HIP, hippocampus; CBL, cerebellum; RB, remaining brain) from 4- to 5-month-old nontransgenic littermate (non-Tg) and Thy1-APPL1 transgenic (Thy1-APPL1) mice. Overexpressed flag-tagged APPL1 proteins were detected in all brain regions in Thy1-APPL1 mice. B , Representative brain section immunolabeling with an anti-APPL1 antibody at low magnification (scale bar, 1 mm) demonstrates the APPL1 overexpression patterns in various brain regions of Thy1-APPL1 mice. C , Representative immunofluorescent images at low (40×) and high (120×) magnification show APPL1 (green) and NeuN (red) with DAPI (blue) staining in cortex layer V (scale bar, 10 µm; yellow arrows indicate neurons, white arrowheads identify non-neuronal cells; genotype as indicated). D , Western blots probed with anti-APPL1 and anti-rab5 antibodies of both non-Tg and Thy1-APPL1 brain homogenate prepared from mice of the indicated ages. E , Western blot analysis quantification showing increased APPL1 in the brain homogenates of Thy1-APPL1 mice compared with non-Tg mice at the indicated ages. ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM.

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: APPL1 transgene expression in the brain. A , Representative Western blot comparing APPL1 expression in various brain regions (CTX, cortex; HIP, hippocampus; CBL, cerebellum; RB, remaining brain) from 4- to 5-month-old nontransgenic littermate (non-Tg) and Thy1-APPL1 transgenic (Thy1-APPL1) mice. Overexpressed flag-tagged APPL1 proteins were detected in all brain regions in Thy1-APPL1 mice. B , Representative brain section immunolabeling with an anti-APPL1 antibody at low magnification (scale bar, 1 mm) demonstrates the APPL1 overexpression patterns in various brain regions of Thy1-APPL1 mice. C , Representative immunofluorescent images at low (40×) and high (120×) magnification show APPL1 (green) and NeuN (red) with DAPI (blue) staining in cortex layer V (scale bar, 10 µm; yellow arrows indicate neurons, white arrowheads identify non-neuronal cells; genotype as indicated). D , Western blots probed with anti-APPL1 and anti-rab5 antibodies of both non-Tg and Thy1-APPL1 brain homogenate prepared from mice of the indicated ages. E , Western blot analysis quantification showing increased APPL1 in the brain homogenates of Thy1-APPL1 mice compared with non-Tg mice at the indicated ages. ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM.

Article Snippet: An additional mouse monoclonal antibody against APPL1 (Novus, catalog #NBP2-46536; RRID:AB_3083472; 1:500) was used for coimmunolabeling with a rabbit polyclonal rab5 antibody (Abcam, catalog #ab218624).

Techniques: Expressing, Western Blot, Transgenic Assay, Immunolabeling, Over Expression, Staining, Two Tailed Test

Early endosome alterations in Thy1-APPL1 mice. A , Representative immunofluorescent images of active, GTP-bound rab5 (rab5-GTP; red) and APPL1 (green) in neurons (layer V, prefrontal cortex) of non-Tg and Thy1-APPL1 mice at 12–13 months of age. Shown are images at lower magnification (40×, merged). The white arrow indicating the neuron shown at higher magnification for individual and merged immunofluorescent signal. Scale bar, 10 µm. B , Quantification of the average number, size, and total area of Rab5-GTP immunolabeled endosomes per cortical neuron in non-Tg versus Thy1-APPL1 mice at the indicated ages. Representative electron microscopy images containing dendritic profiles in layer V of prefrontal cortex ( C ) and hippocampus ( D ) regions and ( E ) quantification of average size and circumference of endosomes in both cortex and hippocampi showing the enlargement in Thy1-APPL1 compared with non-Tg mice aged at 12–13 months (red asterisk indicating endosome; 7 mice each genotype for cortex and 4 mice each genotype for hippocampi were used for quantification. Scale bar, 500 nm). Sixty images/mouse were quantified in E ; for cortex sections, a total of 2,635 endosomes from 14 mice were counted, averaging 188 endosomes per mouse; for hippocampal sections, a total of 1,594 endosomes were counted from 8 mice, with an average of 199 endosomes per mouse. F , Representative immunoelectron microscopy images containing dendritic profiles in layer V of prefrontal cortex labeled with rabbit anti-rab5 and mouse anti-APPL1 followed by gold-conjugated secondary anti-rabbit (6 nm, green arrow) and anti-mouse (10 nm, red arrow) in non-Tg and Thy1-APPL1 mice. Scale bar, 100 nm. G , Percentage of rab5/APPL1 double-positive endosomes versus total rab5-positive endosomes in the dendritic region of the cortex and hippocampus significantly increased in Thy1-APPL1 mice (3 mice per genotype. 35–40 images/mouse were used for counting; for cortex sections, a total of 130 endosomes were counted from 6 mice, with an average of 22 endosomes per mouse; for hippocampal sections, a total of 121 endosomes were counted from 6 mice, with an average of 20 endosomes per mouse). * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Early endosome alterations in Thy1-APPL1 mice. A , Representative immunofluorescent images of active, GTP-bound rab5 (rab5-GTP; red) and APPL1 (green) in neurons (layer V, prefrontal cortex) of non-Tg and Thy1-APPL1 mice at 12–13 months of age. Shown are images at lower magnification (40×, merged). The white arrow indicating the neuron shown at higher magnification for individual and merged immunofluorescent signal. Scale bar, 10 µm. B , Quantification of the average number, size, and total area of Rab5-GTP immunolabeled endosomes per cortical neuron in non-Tg versus Thy1-APPL1 mice at the indicated ages. Representative electron microscopy images containing dendritic profiles in layer V of prefrontal cortex ( C ) and hippocampus ( D ) regions and ( E ) quantification of average size and circumference of endosomes in both cortex and hippocampi showing the enlargement in Thy1-APPL1 compared with non-Tg mice aged at 12–13 months (red asterisk indicating endosome; 7 mice each genotype for cortex and 4 mice each genotype for hippocampi were used for quantification. Scale bar, 500 nm). Sixty images/mouse were quantified in E ; for cortex sections, a total of 2,635 endosomes from 14 mice were counted, averaging 188 endosomes per mouse; for hippocampal sections, a total of 1,594 endosomes were counted from 8 mice, with an average of 199 endosomes per mouse. F , Representative immunoelectron microscopy images containing dendritic profiles in layer V of prefrontal cortex labeled with rabbit anti-rab5 and mouse anti-APPL1 followed by gold-conjugated secondary anti-rabbit (6 nm, green arrow) and anti-mouse (10 nm, red arrow) in non-Tg and Thy1-APPL1 mice. Scale bar, 100 nm. G , Percentage of rab5/APPL1 double-positive endosomes versus total rab5-positive endosomes in the dendritic region of the cortex and hippocampus significantly increased in Thy1-APPL1 mice (3 mice per genotype. 35–40 images/mouse were used for counting; for cortex sections, a total of 130 endosomes were counted from 6 mice, with an average of 22 endosomes per mouse; for hippocampal sections, a total of 121 endosomes were counted from 6 mice, with an average of 20 endosomes per mouse). * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Techniques: Immunolabeling, Electron Microscopy, Immuno-Electron Microscopy, Labeling, Two Tailed Test

Age-related impairments of synaptic plasticity and hippocampal-dependent memory deficit in Thy1-APPL1 mice. A–C , Non-Tg and Thy1-APPL1 mice at 7–9 months of age ( n = 5 for both genotype), input/output relationship plots ( A ), LTP by theta-burst stimulation (TBS) in the Schaffer collateral synapses (CA3-CA1) of hippocampal slices ( B ), and averages of fEPSP slopes at 1, 40, and 80 min following tetanic stimulation ( C ) of the hippocampal slices showing no differences between non-Tg and Thy1-APPL1 mice. D–F , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotype), input/output relationship plots ( D ), LTP induced by TBS in the Schaffer collateral synapses (CA3-CA1) in hippocampal slices ( E ), and averages of fEPSP slopes at 1, 40, and 80 min ( F ) showing significant reduction in Thy1-APPL1 mice ( F (1,236) = 6.696, p = 0.0103 for the slope, linear regression). G–I , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotypes), input/output relationship plots ( G ), LTD induced by low-frequency stimulation (LFS; H ) in hippocampal slices of Thy1-APPL1 ( F (1,127) = 14.83, p = 0.0002 for the intercepts, linear regression), and averages of fEPSP slopes at 50 min following LFS induction ( I ) in the hippocampal slices did not show expected reduction in Thy1-APPL1 mice. J , Recognition index at 3 h after familiarization indicated the memory deficit in Thy1-APPL1 mice at 12–13 months of age ( t (18) = 2.187, p = 0.042). * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Age-related impairments of synaptic plasticity and hippocampal-dependent memory deficit in Thy1-APPL1 mice. A–C , Non-Tg and Thy1-APPL1 mice at 7–9 months of age ( n = 5 for both genotype), input/output relationship plots ( A ), LTP by theta-burst stimulation (TBS) in the Schaffer collateral synapses (CA3-CA1) of hippocampal slices ( B ), and averages of fEPSP slopes at 1, 40, and 80 min following tetanic stimulation ( C ) of the hippocampal slices showing no differences between non-Tg and Thy1-APPL1 mice. D–F , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotype), input/output relationship plots ( D ), LTP induced by TBS in the Schaffer collateral synapses (CA3-CA1) in hippocampal slices ( E ), and averages of fEPSP slopes at 1, 40, and 80 min ( F ) showing significant reduction in Thy1-APPL1 mice ( F (1,236) = 6.696, p = 0.0103 for the slope, linear regression). G–I , Non-Tg and Thy1-APPL1 mice at 12–13 months of age ( n = 5 for both genotypes), input/output relationship plots ( G ), LTD induced by low-frequency stimulation (LFS; H ) in hippocampal slices of Thy1-APPL1 ( F (1,127) = 14.83, p = 0.0002 for the intercepts, linear regression), and averages of fEPSP slopes at 50 min following LFS induction ( I ) in the hippocampal slices did not show expected reduction in Thy1-APPL1 mice. J , Recognition index at 3 h after familiarization indicated the memory deficit in Thy1-APPL1 mice at 12–13 months of age ( t (18) = 2.187, p = 0.042). * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data is shown as mean ± SEM.

Techniques: Two Tailed Test

NGF retrograde signaling and involvement of APPL1 in healthy neurons and after a rise in neuronal APPL1 levels related to Alzheimer's disease in APPL1 overexpressing neurons. Schematic diagram depicts ( A ) the endocytosis of APP, NGF, and its receptor TrkA into a rab5 early endosome. TrkB mediation of BDNF signaling by APPL1 (data not shown) is considered to follow a similar sequence. B , Normal NGF signaling is facilitated by recruitment of APPL1, a direct TrkA ligand and adaptor for other signaling molecules mediating retrograde transport of a maturing endosome carrying the NGF signal to the nucleus to activate a neurotrophic transcriptional program supporting functioning of ChAT neurons and other NGF targets. In AD (not shown; see main text), abnormally elevated APP-βCTF levels arising via multiple possible mechanisms raise levels of the activated form of rab5 (rab5-GTP) on endosomal membranes, in part by recruiting more APPL1 to the endosome via the phosphotyrosine binding (PTB) domain of APPL1 . APPL1's greater affinity for rab5-GTP prolongs association of this activated form on the endosome, thus promoting a pathogenic rab5 hyperactivation leading to increased endocytosis, early endosomal fusion, and endosome enlargement, as depicted in C . C , Moderately elevating APPL1 selectively in neurons of Thy1-APPL1 mice phenocopies mouse models of APP-βCTF elevation or rab5 overexpression with respect to rab5 hyperactivation, abnormal endosome enlargement, stasis of endosome transport, synaptic plasticity deficits, and basal forebrain cholinergic neurodegeneration. These pathological effects, as observed in AD brain, also reflect impaired NGF/TrkA signaling and decreased expression of genes for neuronal survival, growth and differentiation ( ; ). Further information is provided in the text and in more detail in reviews ( ; ).

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: NGF retrograde signaling and involvement of APPL1 in healthy neurons and after a rise in neuronal APPL1 levels related to Alzheimer's disease in APPL1 overexpressing neurons. Schematic diagram depicts ( A ) the endocytosis of APP, NGF, and its receptor TrkA into a rab5 early endosome. TrkB mediation of BDNF signaling by APPL1 (data not shown) is considered to follow a similar sequence. B , Normal NGF signaling is facilitated by recruitment of APPL1, a direct TrkA ligand and adaptor for other signaling molecules mediating retrograde transport of a maturing endosome carrying the NGF signal to the nucleus to activate a neurotrophic transcriptional program supporting functioning of ChAT neurons and other NGF targets. In AD (not shown; see main text), abnormally elevated APP-βCTF levels arising via multiple possible mechanisms raise levels of the activated form of rab5 (rab5-GTP) on endosomal membranes, in part by recruiting more APPL1 to the endosome via the phosphotyrosine binding (PTB) domain of APPL1 . APPL1's greater affinity for rab5-GTP prolongs association of this activated form on the endosome, thus promoting a pathogenic rab5 hyperactivation leading to increased endocytosis, early endosomal fusion, and endosome enlargement, as depicted in C . C , Moderately elevating APPL1 selectively in neurons of Thy1-APPL1 mice phenocopies mouse models of APP-βCTF elevation or rab5 overexpression with respect to rab5 hyperactivation, abnormal endosome enlargement, stasis of endosome transport, synaptic plasticity deficits, and basal forebrain cholinergic neurodegeneration. These pathological effects, as observed in AD brain, also reflect impaired NGF/TrkA signaling and decreased expression of genes for neuronal survival, growth and differentiation ( ; ). Further information is provided in the text and in more detail in reviews ( ; ).

Techniques: Sequencing, Binding Assay, Over Expression, Expressing

Western blot analysis and synaptic vesicle endocytosis (SVE) revealed the abnormalities in hippocampal synaptosomes of Thy1-APPL1 mice. A , Representative Western blots from one of the two experiments showing various protein markers in hippocampal homogenates and hippocampal synaptosome preparations from non-Tg and Thy1-APPL1 mice ( n = 6 each genotype). Quantitation of these Western blots ( B , C ) shows significantly higher levels of APPL1, βCTF, PHF1, and the ratio of PHF1/total tau in synaptosomes of Thy1-APPL1 compared with non-Tg mice at 12–13 months of age. D , Representative fluorescent images of internalized FM4-64 (red) with corresponding CMFDA labeling of total synaptosomes (green) using hippocampal synaptosomes prepared from non-Tg and Thy1-APPL1 mice at 12–13 months of age. E , The ratio of internalized FM4-64 to CMDFA was significantly increased in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months, but not in 7 months of age, indicating elevated endocytosis in older Thy1-APPL1 mice compared with non-Tg. F , Representative images of CMFDA (green) labeling followed by anti-rab5 (red) immunolabeling and ( G ) quantification of the intensity of rab5 to CMFDA showing an increase in rab5 immunosignal per CMFDA-labeled synaptosome in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months of age. Scale bar, 5 µm, * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM. Western blots of hippocampal synaptosome (10 µg of synaptosome protein, input; H ), and GTP-agarose pull-down of hippocampal synaptosome (200 µg of synaptosome protein) from non-Tg and Thy1-APPL1 mice ( I ) probed with anti-APPL1 and anti-rab5 antibodies, total protein stained by Revert 700 Total Protein Stain are also shown at the bottom of H and I . The APPL1 and rab5 band density in both input and pull-down against total protein in relation with non-Tg are presented in J , * p < 0.05, *** p < 0.001, one-way ANOVA. Data is shown as mean ± SEM.

Journal: The Journal of Neuroscience

Article Title: Increased Neuronal Expression of the Early Endosomal Adaptor APPL1 Replicates Alzheimer’s Disease-Related Endosomal and Synaptic Dysfunction with Cholinergic Neurodegeneration

doi: 10.1523/JNEUROSCI.2331-24.2025

Figure Lengend Snippet: Western blot analysis and synaptic vesicle endocytosis (SVE) revealed the abnormalities in hippocampal synaptosomes of Thy1-APPL1 mice. A , Representative Western blots from one of the two experiments showing various protein markers in hippocampal homogenates and hippocampal synaptosome preparations from non-Tg and Thy1-APPL1 mice ( n = 6 each genotype). Quantitation of these Western blots ( B , C ) shows significantly higher levels of APPL1, βCTF, PHF1, and the ratio of PHF1/total tau in synaptosomes of Thy1-APPL1 compared with non-Tg mice at 12–13 months of age. D , Representative fluorescent images of internalized FM4-64 (red) with corresponding CMFDA labeling of total synaptosomes (green) using hippocampal synaptosomes prepared from non-Tg and Thy1-APPL1 mice at 12–13 months of age. E , The ratio of internalized FM4-64 to CMDFA was significantly increased in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months, but not in 7 months of age, indicating elevated endocytosis in older Thy1-APPL1 mice compared with non-Tg. F , Representative images of CMFDA (green) labeling followed by anti-rab5 (red) immunolabeling and ( G ) quantification of the intensity of rab5 to CMFDA showing an increase in rab5 immunosignal per CMFDA-labeled synaptosome in hippocampal synaptosomes of Thy1-APPL1 mice at 12–13 months of age. Scale bar, 5 µm, * p < 0.05, *** p < 0.001, two-tailed, unpaired t test. Data shown as mean ± SEM. Western blots of hippocampal synaptosome (10 µg of synaptosome protein, input; H ), and GTP-agarose pull-down of hippocampal synaptosome (200 µg of synaptosome protein) from non-Tg and Thy1-APPL1 mice ( I ) probed with anti-APPL1 and anti-rab5 antibodies, total protein stained by Revert 700 Total Protein Stain are also shown at the bottom of H and I . The APPL1 and rab5 band density in both input and pull-down against total protein in relation with non-Tg are presented in J , * p < 0.05, *** p < 0.001, one-way ANOVA. Data is shown as mean ± SEM.

Techniques: Western Blot, Quantitation Assay, Labeling, Immunolabeling, Two Tailed Test, Staining

Movement of the Akt adaptor protein APPL1 from the cytosol to the early endosome by endosomal H 2 O 2 during EGF activation. ( A ) Schematic representation of APPL1 domains showing three parts (gray boxes): BAR (essential for Rab5-GTP binding and dimerization), pleckstrin homology (PH, participating in association with Rab5-GTP), and phosphotyrosine binding (PTB, required for binding to activated receptors and Akt). ( B ) Cos7 cells were deprived of serum for 5 h and incubated in a high-glucose medium in the presence of GOx (20 mU/mL) for 10 min. Alternatively, the cells were exposed to GOx for 10 min, washed, and incubated in the absence of GOx for another 30 min (wash out). The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The regions indicated by the arrows are shown at a higher magnification in the second row. ( C ) Quantitative analysis of the relative fluorescence intensity (RFI) from ( B ). Data are presented as means ± SEM ( n = 4 cells for each condition). ** p < 0.01. ( D ) Cos 7 cells were deprived of serum for 5 h, incubated in the absence (Buffer) or presence of DPI (10 μM) for 30 min or catalase (2 mg/mL), and incubated with additional EGF (200 ng/mL) at the selected time points. Selected snapshot confocal microscopy images of the fixed cells were shown for endogenous APPL1. Arrowheads indicate the areas shown at a higher magnification. ( E ) Quantitative analysis of the RFI from ( D ). Data are presented as means ± SEM ( n = 7 cells for each condition). * p < 0.05, ** p < 0.01. ( F ) Cat-Endo- or catalase-expressing Cos7 cells were deprived of serum for 5 h and stimulated with EGF (200 ng/mL) for 1 min. The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The RFI of endosomal APPL1 is presented as means ± SEM ( n = 3 images for each condition). ** p < 0.01. ( G ) HeLa cells were transfected with control siRNA (siCont) or siRNA for APPL1 (siAPPL1) for 72 h and deprived of serum for 5 h. After EGF (200 ng/mL) was added to the cells and left for 1 min, cell lysates were analyzed using antibodies against the proteins listed. ( H ) Quantitative analysis of the relative band intensity of pS 473 Akt and pT 308 Akt from ( G ). Data are presented as means ± SEM ( n = 3 blots). * p < 0.05.

Journal: Antioxidants

Article Title: Endosomal H 2 O 2 Molecules Act as Signaling Mediators in Akt/PKB Activation

doi: 10.3390/antiox14050594

Figure Lengend Snippet: Movement of the Akt adaptor protein APPL1 from the cytosol to the early endosome by endosomal H 2 O 2 during EGF activation. ( A ) Schematic representation of APPL1 domains showing three parts (gray boxes): BAR (essential for Rab5-GTP binding and dimerization), pleckstrin homology (PH, participating in association with Rab5-GTP), and phosphotyrosine binding (PTB, required for binding to activated receptors and Akt). ( B ) Cos7 cells were deprived of serum for 5 h and incubated in a high-glucose medium in the presence of GOx (20 mU/mL) for 10 min. Alternatively, the cells were exposed to GOx for 10 min, washed, and incubated in the absence of GOx for another 30 min (wash out). The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The regions indicated by the arrows are shown at a higher magnification in the second row. ( C ) Quantitative analysis of the relative fluorescence intensity (RFI) from ( B ). Data are presented as means ± SEM ( n = 4 cells for each condition). ** p < 0.01. ( D ) Cos 7 cells were deprived of serum for 5 h, incubated in the absence (Buffer) or presence of DPI (10 μM) for 30 min or catalase (2 mg/mL), and incubated with additional EGF (200 ng/mL) at the selected time points. Selected snapshot confocal microscopy images of the fixed cells were shown for endogenous APPL1. Arrowheads indicate the areas shown at a higher magnification. ( E ) Quantitative analysis of the RFI from ( D ). Data are presented as means ± SEM ( n = 7 cells for each condition). * p < 0.05, ** p < 0.01. ( F ) Cat-Endo- or catalase-expressing Cos7 cells were deprived of serum for 5 h and stimulated with EGF (200 ng/mL) for 1 min. The fixed cells were subjected to immunofluorescence analysis by using the antibodies to APPL1. The RFI of endosomal APPL1 is presented as means ± SEM ( n = 3 images for each condition). ** p < 0.01. ( G ) HeLa cells were transfected with control siRNA (siCont) or siRNA for APPL1 (siAPPL1) for 72 h and deprived of serum for 5 h. After EGF (200 ng/mL) was added to the cells and left for 1 min, cell lysates were analyzed using antibodies against the proteins listed. ( H ) Quantitative analysis of the relative band intensity of pS 473 Akt and pT 308 Akt from ( G ). Data are presented as means ± SEM ( n = 3 blots). * p < 0.05.

Article Snippet: The following substances were used: diphenylene iodonium (DPI, ALX-430-005-M005; Enzo Life Sciences, Farmingdale, NY, USA); epidermal growth factor (EGF, PHG0311; Invitrogen, Carlsbad, CA, USA); glucose oxidase (GOx, 345386; Sigma-Aldrich, St. Louis, MO, USA); 4′,6-diamidino-2-phenylindole (DAPI, 10236276001; Roche, Basel, Switzerland); Alexa-568 conjugated Transferrin ( T23365 ) and Alexa-555 conjugated EGF (E35350; Thermo Fisher Scientific, Waltham, MA, USA); mouse monoclonal antibody to GFP (A11120) and rabbit polyclonal antibody to GFP (A11122; Thermo Fisher Scientific, Waltham, MA, USA); mouse monoclonal antibody to Rictor (ab56578; Abcam, Cambridge, MA, USA); mouse monoclonal antibody to mSIN1 (05-1044) and mouse monoclonal antibody to α-actin (AC-74, A3853; Sigma-Aldrich, St. Louis, MO, USA); rabbit polyclonal antibody to catalase (LF-PA0060; AbFrontier, Seoul, Republic of Korea); rabbit monoclonal antibody to Rab5 (3547), rabbit polyclonal antibody to phospho-Akt (Ser 473 ; 9271), rabbit monoclonal antibody to phospho-Akt (Thr 308 ; 13038), rabbit polyclonal antibody to Akt (9272), and rabbit monoclonal antibody to APPL1 (3858; Cell Signaling Technology, Danvers, MA, USA); and mouse monoclonal antibody to EEA1 (610457; BD Biosciences (Franklin Lakes, NJ, USA).

Techniques: Activation Assay, Binding Assay, Incubation, Immunofluorescence, Fluorescence, Confocal Microscopy, Expressing, Transfection, Control

Proposed model illustrating the signaling role of endosomal H 2 O 2 in Akt activation by the enhanced recruitment of APPL1 and mTorc2 in endosomes. See the for details. EGFR, epidermal growth factor; NOX, NADPH oxidase.

Journal: Antioxidants

Article Title: Endosomal H 2 O 2 Molecules Act as Signaling Mediators in Akt/PKB Activation

doi: 10.3390/antiox14050594

Figure Lengend Snippet: Proposed model illustrating the signaling role of endosomal H 2 O 2 in Akt activation by the enhanced recruitment of APPL1 and mTorc2 in endosomes. See the for details. EGFR, epidermal growth factor; NOX, NADPH oxidase.

Techniques: Activation Assay

Internalized EP2 receptors traffic through very early endosomes (A) Schematic representation of endosomal compartments and their cargo. (B) Representative images from FLAG-βAR and FLAG-EP2 as captured by TIRF microscopy, 5 min post isoproterenol and PGE2 stimulation, respectively. Arrows indicate visible endosomal lumen in FLAG-βAR positive endosomes. Quantitation of endosome diameter (right panel). 182 endosomes (FLAG-EP2) and 264 endosomes (FLAG-βAR) were measured from 4 independent cultures. ∗∗∗∗ denotes a p value of <0.0001 via Student’s t test. (C) FLAG-EP2 (green) and APPL1 (red) positive endosomes with co-localization detected as orange staining, 5 min post PGE2 stimulation (left panels) and quantitation of overlay against the average of all cells (right panel). Arrows indicate FLAG-EP2 and APPL1 positive endosomes. Data are obtained from 30 representative regions chosen at random, n = 3. For B and C, scale bars = 20μm, inset = 10μm.

Journal: iScience

Article Title: Spatial-temporal regulation of the prostanoid receptor EP2 co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

doi: 10.1016/j.isci.2024.111170

Figure Lengend Snippet: Internalized EP2 receptors traffic through very early endosomes (A) Schematic representation of endosomal compartments and their cargo. (B) Representative images from FLAG-βAR and FLAG-EP2 as captured by TIRF microscopy, 5 min post isoproterenol and PGE2 stimulation, respectively. Arrows indicate visible endosomal lumen in FLAG-βAR positive endosomes. Quantitation of endosome diameter (right panel). 182 endosomes (FLAG-EP2) and 264 endosomes (FLAG-βAR) were measured from 4 independent cultures. ∗∗∗∗ denotes a p value of <0.0001 via Student’s t test. (C) FLAG-EP2 (green) and APPL1 (red) positive endosomes with co-localization detected as orange staining, 5 min post PGE2 stimulation (left panels) and quantitation of overlay against the average of all cells (right panel). Arrows indicate FLAG-EP2 and APPL1 positive endosomes. Data are obtained from 30 representative regions chosen at random, n = 3. For B and C, scale bars = 20μm, inset = 10μm.

Article Snippet: Rabbit Anti-APPL1 Monoclonal Antibody , Cell Signaling Technology , Cat# 3856; RRID: AB_2056989.

Techniques: Microscopy, Quantitation Assay, Staining

PGE2, but not relaxin, mediated cAMP signaling are regulated by APPL1 and GIPC (A) RT-qPCR analysis of transcripts for APPL1 (left panel) and GIPC (right panel) following their depletion in EnSCs by siRNA. Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 3). (B) Induction of cAMP after 5-min stimulation with PGE2 (left panel) and relaxin (right panel) following depletion of APPL1 and GIPC by siRNA. Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA, unstimulated controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 6). (C) Schematic representation of experimental procedures used to assess resensitization of EP2 receptors depleted of APPL1 and GIPC (left panel). The cAMP signal from a 5-min PGE2 challenge (2 nd response) following an identical desensitization challenge (1 st response) or control, and ligand washout (right panel). Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA, unstimulated controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 3).

Journal: iScience

Article Title: Spatial-temporal regulation of the prostanoid receptor EP2 co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

doi: 10.1016/j.isci.2024.111170

Figure Lengend Snippet: PGE2, but not relaxin, mediated cAMP signaling are regulated by APPL1 and GIPC (A) RT-qPCR analysis of transcripts for APPL1 (left panel) and GIPC (right panel) following their depletion in EnSCs by siRNA. Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 3). (B) Induction of cAMP after 5-min stimulation with PGE2 (left panel) and relaxin (right panel) following depletion of APPL1 and GIPC by siRNA. Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA, unstimulated controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 6). (C) Schematic representation of experimental procedures used to assess resensitization of EP2 receptors depleted of APPL1 and GIPC (left panel). The cAMP signal from a 5-min PGE2 challenge (2 nd response) following an identical desensitization challenge (1 st response) or control, and ligand washout (right panel). Data from individual patients are color-matched and shown with bar graphs denoting mean values. Differing letters indicate significance from NT siRNA, unstimulated controls ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test, n = 3).

Article Snippet: Rabbit Anti-APPL1 Monoclonal Antibody , Cell Signaling Technology , Cat# 3856; RRID: AB_2056989.

Techniques: Quantitative RT-PCR, Comparison, Control

Loss of APPL1 and GIPC inhibits decidualization (A) Schematic depiction of treatment protocol for RNA sequencing in EnSCs. (B) Relative changes in absorbance from an XTT assay to assess cell viability in untreated EnSCs depleted of APPL1 and GIPC for 4 days. Individual patients are shown as dashed lines, with mean values depicted by the bold lines, n = 3. (C) RT-qPCR analysis of APPL1 (left panel) and GIPC (right panel) 4 days following siRNA depletion. Data from individual patients are color-matched with bar-graphs denoted mean values. Differing letters indicate significant between groups ( p < 0.05) (ANOVA and Tukey’s multiple comparison test, n = 3). (D) Heatmap of differentially expressed genes (Bonferroni correction, p < 0.05) identified from bulk RNA-sequencing between untreated (Day 0) NT siRNA cells and those treated with PGE2/MPA for 4 days. A total of 645 differentially expressed genes were identified (435 upregulated and 210 downregulated) with each gene scaled (z-score) across treatments to show changes with APPL1 and GIPC depletion. Red, blue and white colors represent high, medium and low expression, respectively, as per key. Data are n = 3. (E) Venn diagram depicting the number of differentially expressed genes (Bonferroni correction, p < 0.05) identified when comparing APPL1 and GIPC depleted EnSCs to their treatment-matched (untreated; day 0 or PGE2/MPA treated; day 4) NT siRNA cells. Circles are relative in size to the number of genes. (F) Selected Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed genes (full lists of KEGG enrichment terms are available in <xref ref-type=

Tables S2-5 ). The size of circles is relative to the number of genes in each enrichment term, and the color represents p value calculated as a result of enrichment degree. (G) RT-qPCR analysis showing relative changes in decidual genes IGFBP1 (left panel) and PRL (right panel) following 4-day treatment with 8-bromo-cAMP and MPA (C + M) in EnSCs depleted of APPL1 or GIPC. Data from individual patients are color-matched with bar-graphs denoted mean values. Differing letters indicate significant compared to untreated ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test), n = 3. (H) Summary schematic detailing the rapid recycling and resensitization of EP2 receptors through the VEEs and its contribution to decidualization of EnSCs. " width="100%" height="100%">

Journal: iScience

Article Title: Spatial-temporal regulation of the prostanoid receptor EP2 co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

doi: 10.1016/j.isci.2024.111170

Figure Lengend Snippet: Loss of APPL1 and GIPC inhibits decidualization (A) Schematic depiction of treatment protocol for RNA sequencing in EnSCs. (B) Relative changes in absorbance from an XTT assay to assess cell viability in untreated EnSCs depleted of APPL1 and GIPC for 4 days. Individual patients are shown as dashed lines, with mean values depicted by the bold lines, n = 3. (C) RT-qPCR analysis of APPL1 (left panel) and GIPC (right panel) 4 days following siRNA depletion. Data from individual patients are color-matched with bar-graphs denoted mean values. Differing letters indicate significant between groups ( p < 0.05) (ANOVA and Tukey’s multiple comparison test, n = 3). (D) Heatmap of differentially expressed genes (Bonferroni correction, p < 0.05) identified from bulk RNA-sequencing between untreated (Day 0) NT siRNA cells and those treated with PGE2/MPA for 4 days. A total of 645 differentially expressed genes were identified (435 upregulated and 210 downregulated) with each gene scaled (z-score) across treatments to show changes with APPL1 and GIPC depletion. Red, blue and white colors represent high, medium and low expression, respectively, as per key. Data are n = 3. (E) Venn diagram depicting the number of differentially expressed genes (Bonferroni correction, p < 0.05) identified when comparing APPL1 and GIPC depleted EnSCs to their treatment-matched (untreated; day 0 or PGE2/MPA treated; day 4) NT siRNA cells. Circles are relative in size to the number of genes. (F) Selected Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment of differentially expressed genes (full lists of KEGG enrichment terms are available in Tables S2-5 ). The size of circles is relative to the number of genes in each enrichment term, and the color represents p value calculated as a result of enrichment degree. (G) RT-qPCR analysis showing relative changes in decidual genes IGFBP1 (left panel) and PRL (right panel) following 4-day treatment with 8-bromo-cAMP and MPA (C + M) in EnSCs depleted of APPL1 or GIPC. Data from individual patients are color-matched with bar-graphs denoted mean values. Differing letters indicate significant compared to untreated ( p < 0.05) (ANOVA and Dunnett’s multiple comparison test), n = 3. (H) Summary schematic detailing the rapid recycling and resensitization of EP2 receptors through the VEEs and its contribution to decidualization of EnSCs.

Article Snippet: Rabbit Anti-APPL1 Monoclonal Antibody , Cell Signaling Technology , Cat# 3856; RRID: AB_2056989.

Techniques: RNA Sequencing, XTT Assay, Quantitative RT-PCR, Comparison, Expressing

Journal: iScience

Article Title: Spatial-temporal regulation of the prostanoid receptor EP2 co-ordinates PGE2-mediated cAMP signaling in decidualizing human endometrium

doi: 10.1016/j.isci.2024.111170

Figure Lengend Snippet:

Article Snippet: Rabbit Anti-APPL1 Monoclonal Antibody , Cell Signaling Technology , Cat# 3856; RRID: AB_2056989.

Techniques: Recombinant, Bioassay, Sequencing, Gene Expression, RNA Sequencing, Cell Culture, Software, esiRNA, Negative Control

The alterations of MWT, TWL and CWS and APPL1 protein levels in STZ-induced diabetic rats. (a) The MWT variations in STZ-induced diabetic rats and control rats. (b) The TWL variations in STZ-induced diabetic rats and control rats. (c) The variations of cold pain hypersensitivity in STZ-induced diabetic rats and control rats. (d) The expression of APPL1 in STZ-induced diabetic rats and control rats. The abbreviations for the groups of normal control, diabetes are shown as NC and DM (n = 6 for behavioral tests, n = 4 for Western blotting assay, * P < 0.05 vs. NC group, # P < 0.05 vs. baseline). Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: The alterations of MWT, TWL and CWS and APPL1 protein levels in STZ-induced diabetic rats. (a) The MWT variations in STZ-induced diabetic rats and control rats. (b) The TWL variations in STZ-induced diabetic rats and control rats. (c) The variations of cold pain hypersensitivity in STZ-induced diabetic rats and control rats. (d) The expression of APPL1 in STZ-induced diabetic rats and control rats. The abbreviations for the groups of normal control, diabetes are shown as NC and DM (n = 6 for behavioral tests, n = 4 for Western blotting assay, * P < 0.05 vs. NC group, # P < 0.05 vs. baseline). Data are expressed as the means ± SEM.

Article Snippet: Following the blockade with 5% non-fat milk in phosphate-buffered solution (PBS) solution for 1 h, the membranes were incubated with rabbit monoclonal anti-APPL1 antibody (1:1000; Santa Cruz, USA), rabbit polyclonal anti-mTOR antibody (1:1000; Santa Cruz, USA), rabbit monoclonal anti-phospho-mTOR antibody (p-mTOR, Ser2448, 1:1000; Cell Signaling Technology, USA), rabbit anti-Rab5 (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-Akt (1:1000; Cell Signaling Technology, USA), rabbit monoclonal anti-p-Akt (T308, 1:1000; Cell Signaling Technology, USA), rabbit polyclonal anti-AMPK (Abcam, CA, UK), rabbit polyclonal anti-p-AMPK1 (1:1000, Ser485/Ser491; Affinity Biosciences, OH, USA), or anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (1:1000; Hangzhou Goodhere Biotechnology Co. Ltd., China) overnight at 4°C.

Techniques: Control, Expressing, Western Blot

The localization and distribution of APPL1 in spinal cord in PDN rats. (a) to (f) Double labeling of APPL1 (red) with NeuN (green), GFAP (green), OX-42 (green), CGRP (green), IB-4 (green), and NF200 (green) in spinal dorsal horn in PDN rats, and CON rats. (g) The spinal slices were directly stained with Cy3-conjugated and FITC-conjugated species-specific secondary antibodies as the specificity controls. PDN: painful diabetic neuropathy; CON: normal control.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: The localization and distribution of APPL1 in spinal cord in PDN rats. (a) to (f) Double labeling of APPL1 (red) with NeuN (green), GFAP (green), OX-42 (green), CGRP (green), IB-4 (green), and NF200 (green) in spinal dorsal horn in PDN rats, and CON rats. (g) The spinal slices were directly stained with Cy3-conjugated and FITC-conjugated species-specific secondary antibodies as the specificity controls. PDN: painful diabetic neuropathy; CON: normal control.

Techniques: Labeling, Staining, Control

Effects of APPL1 on the formation of dendritic spine and synapse in STZ-induced diabetic rats. (a) to (c) The immunofluorescent staining for Map2 in control rats, PDN rats, APPL1-deficient PDN rats, and APPL1-overexpressed PDN rats. (d) to (f) The immunofluorescent staining for Bassoon in control rats, PDN rats, APPL1-deficient PDN rats, and APPL1-overexpressed PDN rats. The abbreviations for the groups of normal control (CON), CON + shRNA recombinant lentiviral vector, painful diabetic neuropathy + vacant lentiviral vector (PDN), PDN + shRNA recombinant lentiviral vector and PDN + APPL1 overexpression recombinant lentiviral vector are shown as CON, CON + shRNA, PDN, PDN + shRNA and PDN + OXP. (n = 20, * P < 0.05 vs. control group). Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: Effects of APPL1 on the formation of dendritic spine and synapse in STZ-induced diabetic rats. (a) to (c) The immunofluorescent staining for Map2 in control rats, PDN rats, APPL1-deficient PDN rats, and APPL1-overexpressed PDN rats. (d) to (f) The immunofluorescent staining for Bassoon in control rats, PDN rats, APPL1-deficient PDN rats, and APPL1-overexpressed PDN rats. The abbreviations for the groups of normal control (CON), CON + shRNA recombinant lentiviral vector, painful diabetic neuropathy + vacant lentiviral vector (PDN), PDN + shRNA recombinant lentiviral vector and PDN + APPL1 overexpression recombinant lentiviral vector are shown as CON, CON + shRNA, PDN, PDN + shRNA and PDN + OXP. (n = 20, * P < 0.05 vs. control group). Data are expressed as the means ± SEM.

Techniques: Staining, Control, shRNA, Recombinant, Plasmid Preparation, Over Expression

The double labeling of APPL1 (green) and p-mTOR (red) in control rats and PDN rats. The abbreviations for the groups of control, painful diabetic neuropathy (PDN) are shown as CON, PDN (n = 20, * P < 0.05 vs. control group). Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: The double labeling of APPL1 (green) and p-mTOR (red) in control rats and PDN rats. The abbreviations for the groups of control, painful diabetic neuropathy (PDN) are shown as CON, PDN (n = 20, * P < 0.05 vs. control group). Data are expressed as the means ± SEM.

Techniques: Labeling, Control

Effects of APPL1 genetic knockdown on the expression of p-mTOR and hypersensitivity in STZ-induced diabetic rats. (a) The MWT values in normal control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (b) The TWL values in normal control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (c) The protein expression of APPL1 in spinal dorsal horn in control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (d) The expression of p-mTOR in spinal dorsal horn in control rats, PDN rats, and PDN rats with APPL1 knockdown. The abbreviations for the groups of normal control (CON), CON + shRNA encoding APPL1 (shRNA) painful diabetic neuropathy + vacant lentiviral vector (PDN) and PDN + shRNA are shown as CON, CON+shRNA, PDN and PDN+shRNA (n = 6 for behavioral tests, n = 4 for Western blotting assay, * P < 0.05 vs. CON group, # P < 0.05 vs. PDN). Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: Effects of APPL1 genetic knockdown on the expression of p-mTOR and hypersensitivity in STZ-induced diabetic rats. (a) The MWT values in normal control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (b) The TWL values in normal control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (c) The protein expression of APPL1 in spinal dorsal horn in control rats, PDN rats, and PDN rats infected with shRNA encoding APPL1. (d) The expression of p-mTOR in spinal dorsal horn in control rats, PDN rats, and PDN rats with APPL1 knockdown. The abbreviations for the groups of normal control (CON), CON + shRNA encoding APPL1 (shRNA) painful diabetic neuropathy + vacant lentiviral vector (PDN) and PDN + shRNA are shown as CON, CON+shRNA, PDN and PDN+shRNA (n = 6 for behavioral tests, n = 4 for Western blotting assay, * P < 0.05 vs. CON group, # P < 0.05 vs. PDN). Data are expressed as the means ± SEM.

Techniques: Knockdown, Expressing, Control, Infection, shRNA, Plasmid Preparation, Western Blot

Effects of genetic overexpression of APPL1 on the expression of p-mTOR and hypersensitivity in STZ-induced diabetic rats. (a) The MWT values in control rats, PDN rats, PDN rats infected with APPL1 overexpression, and PDN rats treated with rapamycin (RAP). (b) The TWL values in control rats, PDN rats, PDN rats infected with APPL1 overexpression, and PDN rats treated with rapamycin. (c) The protein expression of APPL1 in spinal dorsal horn in control rats, PDN rats, and PDN rats with APPL1 overexpression. (d) The expression of p-mTOR in spinal dorsal horn in control rats, PDN rats, PDN rats with APPL1 overexpression, and PDN rats treated with rapamycin. (e) The expression of p-S6K in spinal dorsal horn in control rats, PDN rats, and PDN rats treated with rapamycin. The abbreviations for the groups of normal control, painful diabetic neuropathy (PDN) + vacant lentiviral vector, PDN + APPL1 genetic overexpression and PDN + rapamycin are shown as CON, PDN, OXP and RAP. (n = 6 for behavioral tests, n = 4 for Western blotting assay; * P < 0.05 vs. CON group, # P < 0.05 vs. PDN group in ; * P < 0.05 vs. PDN group in . Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: Effects of genetic overexpression of APPL1 on the expression of p-mTOR and hypersensitivity in STZ-induced diabetic rats. (a) The MWT values in control rats, PDN rats, PDN rats infected with APPL1 overexpression, and PDN rats treated with rapamycin (RAP). (b) The TWL values in control rats, PDN rats, PDN rats infected with APPL1 overexpression, and PDN rats treated with rapamycin. (c) The protein expression of APPL1 in spinal dorsal horn in control rats, PDN rats, and PDN rats with APPL1 overexpression. (d) The expression of p-mTOR in spinal dorsal horn in control rats, PDN rats, PDN rats with APPL1 overexpression, and PDN rats treated with rapamycin. (e) The expression of p-S6K in spinal dorsal horn in control rats, PDN rats, and PDN rats treated with rapamycin. The abbreviations for the groups of normal control, painful diabetic neuropathy (PDN) + vacant lentiviral vector, PDN + APPL1 genetic overexpression and PDN + rapamycin are shown as CON, PDN, OXP and RAP. (n = 6 for behavioral tests, n = 4 for Western blotting assay; * P < 0.05 vs. CON group, # P < 0.05 vs. PDN group in ; * P < 0.05 vs. PDN group in . Data are expressed as the means ± SEM.

Techniques: Over Expression, Expressing, Control, Infection, Plasmid Preparation, Western Blot

The involvement of AMPK and Akt in the negative regulation of mTOR by APPL1 in STZ-induced diabetic rats. The variations of p-AMPK and p-Akt in normal control rats with vacant lentiviral vector control, normal control rats with APPL1 knockdown, PDN rats, and PDN rats with APPL1 knockdown, and PDN rats with APPL1 overexpression. The abbreviations for the groups of negative control, normal control + shRNA encoding APPL1, painful diabetic neuropathy (PDN), PDN + shRNA encoding APPLL (shRNA) and PDN + APPL1 genetic overexpression are shown as CON, CON + shRNA, PDN, PDN + shRNA, and PDN + OXP. (n = 4, * P < 0.05 vs. NC group, # P < 0.05 vs. PDN group). Data are expressed as the means ± SEM.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: The involvement of AMPK and Akt in the negative regulation of mTOR by APPL1 in STZ-induced diabetic rats. The variations of p-AMPK and p-Akt in normal control rats with vacant lentiviral vector control, normal control rats with APPL1 knockdown, PDN rats, and PDN rats with APPL1 knockdown, and PDN rats with APPL1 overexpression. The abbreviations for the groups of negative control, normal control + shRNA encoding APPL1, painful diabetic neuropathy (PDN), PDN + shRNA encoding APPLL (shRNA) and PDN + APPL1 genetic overexpression are shown as CON, CON + shRNA, PDN, PDN + shRNA, and PDN + OXP. (n = 4, * P < 0.05 vs. NC group, # P < 0.05 vs. PDN group). Data are expressed as the means ± SEM.

Techniques: Control, Plasmid Preparation, Knockdown, Over Expression, Negative Control, shRNA

Effects of APPL1 genetic knockdown or overexpression on Rab5 expression in STZ-induced diabetic rats. (a) Effects of APPL1 knockdown on Rab5 expression in STZ-induced diabetic rats. (b) Effects of APPL1 genetic overexpression on Rab5 expression in STZ-induced diabetic rats. (c) to (d) Immunostaining for Rab5 in lumbar spinal dorsal horn in APPL1 genetic knockdown or overexpression diabetic rats. The upper panel shows the immunoactivity of Rab5 in lumbar spinal dorsal horn of diabetic rats; the lower bars show the levels of Rab5 by quantifying the gray value of Rab5 immunostaining images. The abbreviations for the groups of normal control, painful diabetic neuropathy (PDN), PDN + APPL1 genetic knockdown, and PDN + APPL1 genetic overexpression are shown as CON, PDN, shRNA, and OXP, respectively (n = 20 for immunofluorescent staining assay, n = 4 for Western blotting assay, * P < 0.05 vs. PDN group in ; * P < 0.05 vs. CON group, # P < 0.05 vs. PDN group in . Data are expressed as the means ± SEM. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Journal: Molecular Pain

Article Title: mTOR activation due to APPL1 deficiency exacerbates hyperalgesia via Rab5/Akt and AMPK signaling pathway in streptozocin-induced diabetic rats

doi: 10.1177/1744806919880643

Figure Lengend Snippet: Effects of APPL1 genetic knockdown or overexpression on Rab5 expression in STZ-induced diabetic rats. (a) Effects of APPL1 knockdown on Rab5 expression in STZ-induced diabetic rats. (b) Effects of APPL1 genetic overexpression on Rab5 expression in STZ-induced diabetic rats. (c) to (d) Immunostaining for Rab5 in lumbar spinal dorsal horn in APPL1 genetic knockdown or overexpression diabetic rats. The upper panel shows the immunoactivity of Rab5 in lumbar spinal dorsal horn of diabetic rats; the lower bars show the levels of Rab5 by quantifying the gray value of Rab5 immunostaining images. The abbreviations for the groups of normal control, painful diabetic neuropathy (PDN), PDN + APPL1 genetic knockdown, and PDN + APPL1 genetic overexpression are shown as CON, PDN, shRNA, and OXP, respectively (n = 20 for immunofluorescent staining assay, n = 4 for Western blotting assay, * P < 0.05 vs. PDN group in ; * P < 0.05 vs. CON group, # P < 0.05 vs. PDN group in . Data are expressed as the means ± SEM. GAPDH: glyceraldehyde 3-phosphate dehydrogenase.

Techniques: Knockdown, Over Expression, Expressing, Immunostaining, Control, shRNA, Staining, Western Blot

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