Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 1. Expression of FKBP51 and SYP in PTZ and KA epileptic mouse models. (A,B) Expression of FKBP51 in PTZ and KA epileptic mouse models. (C–F) Expression of “total SYP and PSD95” and “SYP and PSD95 after extraction of synaptosomes” in the

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Expressing, Extraction

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 2. Localization of FKBP51 in epileptic brain tissue detected by immunofluorescence FKBP51 was expressed in the CA1 in the hippocampus of epileptic mice. White arrows indicate co-expression with GFAP and NeuN (n = 5). The scale bar represents 50

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Immunofluorescence, Expressing

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 3. Injection site and the selection of AAV-FKBP51-shRNA sequences. (A) According to the mouse atlas, the injection site is bilat-

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Injection, Selection, shRNA

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 4. Effects of FKBP51 on seizure grade and seizure latency in mice. (A,B) The seizure grades and latent period in the PTZ+AAV-

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques:

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 5. Effect of endogenous FKBP51 inhibition on apical dendritic spines in CA1 of the hippocampus. (A) The distribution of apical dendritic spines in the hippocampal CA1 and local enlarged images of the Con group, Model group, PTZ+AAV-GFP group, and

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Inhibition

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 6. Effect of endogenous FKBP51 inhibition on synaptic ultrastructure. (A) Clear synaptic structure (selected from Con group),

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Inhibition

Journal: Journal of Integrative Neuroscience

Article Title: FKBP51 is Involved in Epileptic Seizure by Regulating PSD95 in a PTZ-Induced Epileptic Mouse Model

doi: 10.31083/jin25710

Figure Lengend Snippet: Fig. 7. Expression of PSD95 and SYP after decreasing the expression of FKBP51. (A–D) “Total PSD95 and SYP” and “PSD95

Article Snippet: FKBP51 rabbit antibodies (1:1000, 14155-1-AP, Proteintech Group, Inc, Wuhan, China), PSD95 rabbit antibodies (1:5000, 20665-1-AP, Proteintech Group, Inc, Wuhan, China), SYP rabbit antibodies (1:40,000, Cat No. 17785-1-AP, Proteintech Group, Inc., Wuhan, China), GAPDH rabbit antibodies (1:10,000, 10494-1-AP, Proteintech Group, Inc, Wuhan, China), and β-tubulin mouse antibodies (1:50,000, 66240-1-Ig, Proteintech Group, Inc, Wuhan, China) were added and incubated overnight on a shaking table at 4 °C.

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Differential Dynamics and Roles of FKBP51 Isoforms and Their Implications for Targeted Therapies

doi: 10.3390/ijms252212318

Figure Lengend Snippet: FKBP5/51 transcription variants and isoforms: ( a ) Schematic view of the FKBP5 locus on human chromosome 6 and the four splicing variants of the gene (adapted from gtexportal.org ). ( b ) Schematic view of FKBP51 isoform 1 and 2 protein structures and 3D structure models generated with the Swiss model repository server of the expasy portal ( swissmodel.expasy.org (accessed on 16 October 2024); ). Domains are indicated in black and experimentally validated domain-associated binding partners in blue. ( c ) Transcription variant-specific FKBP5 expression throughout human tissues (adapted from gtexportal.org ; ). The data used for the analyses described in this figure were obtained from: www.gtexportal.org , the GTEx Portal on 14 September 2023. The terms and conditions for the use of data and images can be found here: https://www.gtexportal.org/home/downloads/adult-gtex/overview , accessed on 14 September 2023.

Article Snippet: The following primary antibodies were used for Western blot: BECN1 (1:1000, #3495), ATG12 (1:1000, #2010), LC3B-II/I (1:1000, #2775Cell), AKT (1:1000, #4691), and pAKT (Ser473 1:1000, #4058 and #9275) were acquired from Cell Signaling, Danver, MA, USA; FKBP51 (1:1000, A301-430A, Bethyl, Montgomery, TX, USA, and ab46002, Abcam, Cambridge, UK), FKBP51 specific for isoform 2 (generously provided by the Maria-Fiammetta Romano lab, Federico II University, Naples, Italy); Actin (1:5000, sc-1616, Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH (1:8000, CB1001 Millipore, Burlington, MA, USA).

Techniques: Generated, Binding Assay, Variant Assay, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Differential Dynamics and Roles of FKBP51 Isoforms and Their Implications for Targeted Therapies

doi: 10.3390/ijms252212318

Figure Lengend Snippet: Expression of FKBP5 splicing variants in HeLa cells: ( a ) RT-qPCR quantification of FKBP51 variants in unstimulated HeLa cells. ( b ) RT-qPCR quantification of FKBP51 variants, expressed as fold change in Dex-treated over vehicle-treated, normalized on the housekeeper YWHAZ of HeLa cells treated with 100 nM Dex or vehicle for 24 h. Two-way ANOVA with Geisser–Greenhouse correction (shown in the box) and Sidak’s multiple comparisons test (shown in the graph). Data shown as mean ± SEM. ( c ) Fold change in FKBP5 variants 1 and 4 over vehicle and normalized over YWHAZ at 0, 1, 3, 6, and 23 h after Dex stimulation. Mixed effects model with Geisser–Greenhouse correction (shown in the box) and Sidak’s multiple comparisons test (shown in the graph). Data shown as box-and-whisker plot (Tukey style). ( d ) Pulse-chase assay of FKBP51 isoform 1 and 2 of HeLa cells transfected with HaloTag ® -tagged-isoform 1 or HaloTag ® -tagged-isoform 2, pulsed with a fluorophore, and chased for 2, 4, 8, and 16 h. Dotted line indicates half-life of the protein. Quantifications were made from Western blots. * p < 0.05. Two-way ANOVA (shown in the box) and Sidak’s multiple comparisons test (shown in the graph). Data shown as mean ± SEM. For all statistics * p < 0.05, ** p < 0.01, **** p < 0.0001, ns = not significant.

Article Snippet: The following primary antibodies were used for Western blot: BECN1 (1:1000, #3495), ATG12 (1:1000, #2010), LC3B-II/I (1:1000, #2775Cell), AKT (1:1000, #4691), and pAKT (Ser473 1:1000, #4058 and #9275) were acquired from Cell Signaling, Danver, MA, USA; FKBP51 (1:1000, A301-430A, Bethyl, Montgomery, TX, USA, and ab46002, Abcam, Cambridge, UK), FKBP51 specific for isoform 2 (generously provided by the Maria-Fiammetta Romano lab, Federico II University, Naples, Italy); Actin (1:5000, sc-1616, Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH (1:8000, CB1001 Millipore, Burlington, MA, USA).

Techniques: Expressing, Quantitative RT-PCR, Whisker Assay, Pulse Chase, Transfection, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Differential Dynamics and Roles of FKBP51 Isoforms and Their Implications for Targeted Therapies

doi: 10.3390/ijms252212318

Figure Lengend Snippet: Differential pathway regulation of FKBP51 isoforms: ( a ) Epifluorescent and bright field imaging of HeLa cells transfected with GFP-control vector, GFP-tagged FKBP51 isoform 1 or GFP-tagged FKBP51 isoform 2 24 h prior to imaging. ( b , c ) GRE-driven reporter gene assay performed in HeLa cells transfected with ( b ) FKBP51 isoform 1, FKBP51 isoform 2 or an empty vector (ctr vector), or ( c ) in WT (expressing both isoform), full KO and Isoform 1 KO (iso1 KO) HeLa cells treated with 0.1 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, or vehicle for 4 h. Two-way ANOVA (shown in the box) with Tukey multiple comparisons test (shown in the graph). * indicates comparison with control/WT and isoform 1/full KO, # indicates comparison between isoform 1 and isoform 2, and $ refers to comparison between WT and iso 1 KO. (** & ## & $$ p < 0.01, *** & ### p < 0.0005, **** & #### p < 0.0001, ns = not significant). ( d ) Representative Western blots for different pathway markers performed on lysates from HeLa cells transfected with FKBP51 isoform 1, FKBP51 isoform 2 or an empty vector ( e – j ) Quantification of Western blots analyses displayed in ( d ): ( e ) phosphorylated AKT (pAKT) normalized on total AKT, ( f – h ) autophagy markers, BECN1, ATG12 and LC3B-II/I; ( i ) phosphorylated DNMT (pDNMT) normalized on total DNMT; ( j ) phosphorylated NFAT (pNFAT) normalized on total NFAT from Jurkat cells transfected with FKBP51 isoform 1, FKBP51 isoform 2 or an empty vector; * p < 0.05, ** p < 0.01, *** p < 0.0005, ns = not significant. Mann–Whitney test. Data shown as mean ± SEM.

Article Snippet: The following primary antibodies were used for Western blot: BECN1 (1:1000, #3495), ATG12 (1:1000, #2010), LC3B-II/I (1:1000, #2775Cell), AKT (1:1000, #4691), and pAKT (Ser473 1:1000, #4058 and #9275) were acquired from Cell Signaling, Danver, MA, USA; FKBP51 (1:1000, A301-430A, Bethyl, Montgomery, TX, USA, and ab46002, Abcam, Cambridge, UK), FKBP51 specific for isoform 2 (generously provided by the Maria-Fiammetta Romano lab, Federico II University, Naples, Italy); Actin (1:5000, sc-1616, Santa Cruz Biotechnology, Dallas, TX, USA), GAPDH (1:8000, CB1001 Millipore, Burlington, MA, USA).

Techniques: Imaging, Transfection, Control, Plasmid Preparation, Reporter Gene Assay, Expressing, Comparison, Western Blot, MANN-WHITNEY

Journal: Nature Communications

Article Title: The AKT2/SIRT5/TFEB pathway as a potential therapeutic target in non-neovascular AMD

doi: 10.1038/s41467-024-50500-z

Figure Lengend Snippet: a Western blot showing elevated levels of FKBP51 and Snap23 in AKT2 overexpressing ARPE19 cells, which were reduced upon simultaneous upregulation of SIRT5 or overexpression of an AKT2 inactive mutant (K14A/R25E). n (biological replicate) = 4. b Immunofluorescence assay showing association of LC3-positive autophagosomes (green) with lysosomes (Lamp1-positive; red) in control ARPE19 cells (arrows in b ), which was significantly reduced upon AKT2 overexpression. Scale bar = 50 μm. n (biological replicate) = 8. c RNAseq analysis from RPE cells of 4-month-old WT and Akt2 KI mice showing differential expression of several autophagy-related genes. n = 3. d Western blot showing reduced expression of autophagy mediators ATG9B and ULK1, as well as upregulation of the autophagosome marker p62/SQSTM1 in Akt2 KI RPE cells, relative to WT. n (biological replicate) = 4. e Chromatin immunoprecipitation showing diminished binding of TFEB on Atg9b promoter region in Akt2 KI RPE cells, compared to WT. n (biological replicate) = 4. f Western blot showing reduced autophagy flux (Ratio of LC3-II/ Vinculin in BafA1 treated vs untreated) in Akt2 KI RPE explants compared to WT when treated with Bafilomycin A1 (BafA1; 1 μm) for 4 h. n (biological replicate) = 4. g ARPE19 cells were transfected with AKT2 construct for 48 h or left untreated (control), followed by an overnight infection with an Adenovirus-GFP-RFP-LC3B construct to label the autophagosomes (yellow) and autolysosomes (red). The number of autolysosomes (red puncta) was significantly decreased in AKT2 overexpressing cells (arrows in c ) when compared to controls, suggesting a decline in autophagy flux. Scale bar = 50 μm. n (biological replicate) = 4. h Transmission electron micrographs showing double membranous autophagosomes in the RPE cells of 15-month-old Akt2 KI mice, but not in age-matched WT. Scale bar = 600 nm. n = 5. All values are Mean ± S.D. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. The statistical tests used in ( a , c, and f ) is One-way ANOVA followed by Tukey’s post-hoc test whereas in ( b , d , g ) is Student’s t-test (Two-sided). The exact p-values are a FKBP51: P = 0.0007 (AKT2-GFP-pcDNA vs untr a nsfected), P = 0.0327 (AKT2-GFP-pcDNA+SIRT-HA-pcDNA vs AKT2-GFP-pcDNA), P = 0.0081 (AKT2(K14A/R25E)-GFP-pcDNA vs AKT2-GFP-pcDNA); b P = 1.598E-10 (AKT2-pcDNA vs Control); d ATG9B: P = 0.0139, p62: P = 0.0013, ULK1: P = 0.017; e P = 0.001 ( Akt2 KI vs WT); f P = 9.45202E-05 ( Akt2 KI vs WT); g Autophagosome: P = 0.0012, Autolysosome: P = 0.000082 (AKT2-pcDNA vs Control). Source Data is provided in the Source Data file.

Article Snippet: The primary antibodies AKT2 (3063S), p-AKT2 (8599S), SIRT5 (8782S), CTSD (69854S), ULK1 (8054T), FKBP51 (8245S), p-TFEB (37681S), and GFP (2555S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Over Expression, Mutagenesis, Immunofluorescence, Control, Quantitative Proteomics, Expressing, Marker, Chromatin Immunoprecipitation, Binding Assay, Transfection, Construct, Infection, Transmission Assay