Journal: Appetite

Article Title: Moderate High Fat Diet Increases Sucrose Self-Administration In Young Rats

doi: 10.1016/j.appet.2012.09.021

Figure Lengend Snippet: Experimental Protocols

Article Snippet: Primary antibodies used were rabbit anti-cFos (1:500) (sc-52) and goat anti-AGRP (1:100) (18634) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques:

Journal: Appetite

Article Title: Moderate High Fat Diet Increases Sucrose Self-Administration In Young Rats

doi: 10.1016/j.appet.2012.09.021

Figure Lengend Snippet: Activation of AGRP neurons at the onset of sucrose self-administration. 3a. Co- localization of cFos and AGRP in arcuate nucleus neuron, 60x magnification. 3b. Number of activated (cFos-immunopositive) AGRP-immunopositive neurons in the mediobasal hypothalamus of peri-pubertal rats trained to self-administer sucrose (n=10), or behavioral controls (handled only; n=6). Both the total number of activated AGRP neurons (left bars) or the number of activated AGRP neurons as a percent of total AGRP neurons (Total = AGRP-positive with or without cFos-immunopositive co-staining) are significantly increased in the rats trained to self-administer sucrose. Timing of euthanasia would reflect activation of the AGRP in anticipation of, or at the start of, the actual self-administration session.

Article Snippet: Primary antibodies used were rabbit anti-cFos (1:500) (sc-52) and goat anti-AGRP (1:100) (18634) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).

Techniques: Activation Assay, Staining

Journal: International Journal of Molecular Sciences

Article Title: Small Heat Shock Protein 22 Improves Cognition and Learning in the Tauopathic Brain

doi: 10.3390/ijms23020851

Figure Lengend Snippet: Hsp22 levels were significantly upregulated by AAV9. ( a ) Timeline of in vivo study. ( b ) Representative images of GFP, wtHsp22, and mtHsp22 levels in non-transgenic (Non-Tg) mice. ( c ) Representative images of GFP, wtHsp22, and mtHsp22 levels in rTg4510 mice. Scale bar represents 200 µm; inset represents 20 µm. ( d ) Quantification of immunohistochemical staining of Hsp22 levels in non-Tg mice (mean ± SEM, * p < 0.05) and ( e ) rTg4510 mice (mean ± SEM, * p < 0.05, ** p < 0.01) GFP ( n = 5/Non-Tg; n = 7/rTg4510), wtHsp22 ( n = 7/Non-Tg; n = 6/rTg4510) mtHsp22 ( n = 6/Non-Tg; n = 6/rTg4510). ( f ) Quantification of Hsp22 levels in non-Tg mice by parallel reaction monitoring (mean ± SEM, * p < 0.05, ** p < 0.01). ( g ) Quantification of Hsp22 levels in rTg4510 mice by quantitative mass spectrometry parallel reaction monitoring (mean ± SEM, ** p < 0.05). GFP ( n = 6/Non-Tg; n = 6/rTg4510), wtHsp22 ( n = 7/Non-Tg; n = 6/rTg4510) mtHsp22 ( n = 6/Non-Tg; n = 6/rTg4510).

Article Snippet: Immunostaining was performed using the following antibodies: rabbit anti-Hsp22 (1:300; Stress Marq, Victoria, BC, Canada; SPC181D), rabbit anti-human tau (1:100,000; Agilent; A002401-2), rabbit anti-pS262 tau (1:1000; AnaSpec; 54973), rabbit anti-pT231(1:300; AnaSpec; 55313), rabbit anti-p62 (1:300; Cell Signaling; 5114S), rabbit anti-T22 (1:15,000; Millipore; ABN454), rabbit anti-doublecortin (DCX; 1:1000; Cell Signaling; 4604S) and developed using 1.4 mM diaminobenzidine and nickel with 0.03% H 2 O 2 following incubation with biotinylated goat anti-rabbit secondary antibody (1:3000; Southern Biotech, Birmingham AL, USA; 4050-08) and Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA; PK-4000).

Techniques: In Vivo, Transgenic Assay, Immunohistochemical staining, Staining, Mass Spectrometry

Journal: International Journal of Molecular Sciences

Article Title: Small Heat Shock Protein 22 Improves Cognition and Learning in the Tauopathic Brain

doi: 10.3390/ijms23020851

Figure Lengend Snippet: Hsp22 overexpression increases synaptic plasticity in rTg4510 mice. After recording a 20-min baseline, long-term potentiation (LTP) was induced in the Schaffer collaterals with high-frequency stimulation (HFS) (5 bursts of 200 Hz separated by 200 ms, repeated 6 times with an intertrain interval of 10 s). fEPSP was recorded for 60 min in ( a ) rTg4510, and ( b ) non-transgenic (Non-Tg) mice injected with GFP ( n = 4/Non-Tg; n = 4/rTg4510), wtHsp22 ( n = 4/Non-Tg; n = 4/rTg4510), or mtHsp22 ( n = 4/Non-Tg; n = 4/rTg4510) (mean ± SEM, * p < 0.05, **** p < 0.0001). Representative traces are shown: 1 (teal) indicates baseline, 2 (pink) indicates early LTP potentiation in the first 3 min following HFS, and 3 (blue) indicates late LTP in the last 3 min of recording. The input/output curves of the fEPSP slope (mV/ms) versus the fiber volley amplitude (mV) in ( c ) rTg4510 and ( d ) Non-Tg mice injected with GFP, wtHsp22, or mtHsp22 (mean ± SEM).

Article Snippet: Immunostaining was performed using the following antibodies: rabbit anti-Hsp22 (1:300; Stress Marq, Victoria, BC, Canada; SPC181D), rabbit anti-human tau (1:100,000; Agilent; A002401-2), rabbit anti-pS262 tau (1:1000; AnaSpec; 54973), rabbit anti-pT231(1:300; AnaSpec; 55313), rabbit anti-p62 (1:300; Cell Signaling; 5114S), rabbit anti-T22 (1:15,000; Millipore; ABN454), rabbit anti-doublecortin (DCX; 1:1000; Cell Signaling; 4604S) and developed using 1.4 mM diaminobenzidine and nickel with 0.03% H 2 O 2 following incubation with biotinylated goat anti-rabbit secondary antibody (1:3000; Southern Biotech, Birmingham AL, USA; 4050-08) and Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA; PK-4000).

Techniques: Over Expression, Transgenic Assay, Injection

Journal: International Journal of Molecular Sciences

Article Title: Small Heat Shock Protein 22 Improves Cognition and Learning in the Tauopathic Brain

doi: 10.3390/ijms23020851

Figure Lengend Snippet: Hsp22 overexpression protects neurons in rTg4510 mice. ( a ) Immunohistochemistry staining and ( b ) quantification of neurons (NeuN/Cresyl violet) in the hippocampi of rTg4510 mice injected with GFP ( n = 6), wtHsp22 ( n = 6), or mtHsp22 ( n = 6) (mean ± SEM; scale bar represents 200 µm; inset represents 10 µm). * p < 0.05.

Article Snippet: Immunostaining was performed using the following antibodies: rabbit anti-Hsp22 (1:300; Stress Marq, Victoria, BC, Canada; SPC181D), rabbit anti-human tau (1:100,000; Agilent; A002401-2), rabbit anti-pS262 tau (1:1000; AnaSpec; 54973), rabbit anti-pT231(1:300; AnaSpec; 55313), rabbit anti-p62 (1:300; Cell Signaling; 5114S), rabbit anti-T22 (1:15,000; Millipore; ABN454), rabbit anti-doublecortin (DCX; 1:1000; Cell Signaling; 4604S) and developed using 1.4 mM diaminobenzidine and nickel with 0.03% H 2 O 2 following incubation with biotinylated goat anti-rabbit secondary antibody (1:3000; Southern Biotech, Birmingham AL, USA; 4050-08) and Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA; PK-4000).

Techniques: Over Expression, Immunohistochemistry, Staining, Injection

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 1. Co-localization of RPS27A/MT2A/HMOX1/UBB and HSPC238 in 293T and SMMC7721 cells. 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag together with either pcDNA3.1-HMOX1-6xHis, pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. The co-localization of HSPC238 with (A) RPS27A, (B) MT2A, (C) HMOX1 and (D) UBB, was visualized on a confocal microscope with anti-His and anti-Flag antibodies. The blue fluorescence is the nuclei stained with DAPI, the green fluorescence is the His-tagged target protein and the red fluorescence is Flag-tagged HSPC238.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Microscopy, Fluorescence, Staining

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 2. HMOX1, MT2A, RPS27A and UBB co-immunoprecipitate with HSPC238. (A) 293T or SMMC7721 cells were transfected with pcDNA3.1, pcDNA3.1-HSPC238-Flag or pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for immunoblotting of His and Flag tag. (B) 293T or SMMC7721 cells were transfected with pcDNA3.1, pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total proteins were extracted for immunoblotting of the His tag. (C) 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag, or pcDNA3.1-HSPC238-Flag together with pcDNA3.1-HMOX1-6xHis pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total proteins were extracted for co-immu- noprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (IB) with anti-His tag, anti-HSPC238, anti-HMOX-1, anti-RPS27A, anti-UBB and anti-MT2A antibodies. (D) 293T or SMMC7721 cells were co-transfected with pcDNA3.1-HSPC238-Flag or pcDNA3.1-HSPC238-Flag together with pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for co-immunoprecipitation (IP) with anti-Flag antibodies, followed by immunoblotting (IB) with anti-His tag, HSPC238, HMOX-1, RPS27A, UBB and MT2A antibodies.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Western Blot, FLAG-tag, Immunoprecipitation

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 3. HSPC238 pull-down by HMOX-1, RPS27A, UBB and MT2A. (A) 293T or SMMC7721 cells were transfected with pcDNA3.1-HSPC238-Flag, alone or in combination with pcDNA3.1-HMOX1-6xHis. Total proteins were extracted for simultaneous purification by nickel column. Three eluate samples with higher concentrations were subjected to western blotting. (B) 293T or SMMC7721 cells were transfected with pcDNA3.1-HSPC238-Flag, alone or in combina- tion with pcDNA3.1-RPS27A-6xHis, pcDNA3.1-UBB-6xHis or pcDNA3.1-MT2A-6xHis. Total protein was extracted for simultaneous purification by nickel column. The eluate sample with the highest concentration as well as cell lysates were subjected to western blotting.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques: Transfection, Purification, Nickel Column, Western Blot, Concentration Assay

Journal: Oncology reports

Article Title: Screening and verification of proteins that interact with HSPC238.

doi: 10.3892/or.2015.4289

Figure Lengend Snippet: Figure 4. A possible mechanism of HSPC238 interaction with HMOX-1, MT2A, RPS27a and UBB.

Article Snippet: Anti-HSPC238 rabbit antibody was obtained from Proteintech Group Inc. (Chicago, IL, USA); anti-HMOX1, antiMT2A, anti-RPS27A and anti-ubiquitin B (UBB) antibodies were purchased from Abclonal (Cambridge, MA, USA); and anti-HIS mouse antibody and anti-Flag mouse antibody were purchased from Zoonbio Tech Co., Ltd. (Nanjing, China).

Techniques:

Journal: Neural Regeneration Research

Article Title: FUBP3 mediates the amyloid-β-induced neuronal NLRP3 expression

doi: 10.4103/NRR.NRR-D-23-01799

Figure Lengend Snippet: NLRP3 is expressed in neurons and regulates tau phosphorylation. (A) Brain sections from 3-month-old wild-type mice were costained with antibodies to NLRP3 (red, Alexa Fluor 564) and the microglial marker Iba1 (green, Alexa Fluor 488). (B) Brain sections from 8-month-old APP/PS1ΔE9 AD model mice were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-Iba1 (green, Alexa Fluor 488) antibodies. Arrowheads indicate NLRP3-positive microglia, and the neuritic plaque is circled. (C) Brain sections from 8-month-old APP/PS1ΔE9 AD model mice were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-NeuN (green, Alexa Fluor 488) antibodies. The arrowhead indicates an NLRP3-positive neuron. (D) Hydrogen peroxide (H 2 O 2 ) was injected into the hippocampus of APP/PS1ΔE9 mice, and brain sections from these mice were costained with antibodies to NLRP3 and the neuronal marker NeuN. Arrowheads indicate NeuN-positive, NLRP3-negative neurons in the peri-injection area. The dashed boxes indicate the areas that are enlarged in the lower panels. (E) Western blot analysis of lysates from the microglial cell line BV2 and primary neurons from wild-type mice (PN WT ). (F) PN WT from wild-type mice and cultured primary neurons from APP Swedish mutant transgenic mice (PN APP ) expressing human APP with the Swedish mutation were costained with anti-NLRP3 (red, Alexa Fluor 564) and anti-NeuN (green, Alexa Fluor 488) antibodies. NLRP3 was not clearly detectable beyond the background staining in PN WT , whereas the NLRP3 signal in PN APP was markedly stronger than in nonneuronal cells. Asterisks indicate nonneuronal (NeuN-negative) cells. (G) Western blot for NLRP3 in Neuro2A cell lysates. (H) Total RNA was extracted from Neuro2A cells and reverse transcribed, and the indicated genes were PCR-amplified. RNA was used as negative control template. (I) Mouse brains were lysed and treated with or without λ-protein phosphatase. The treated lysates were subjected to western blot for p-tau181 and p-tau202/205 to determine the specificity of the antibodies. PN APP were treated with the NLRP3 inhibitors CY-09 and CORM3 for 4 hours. The cell lysates were subjected to western blot for p-tau181 and p-tau202-205. Data are expressed as mean ± SD ( n = 3 independent repeats). * P < 0.05, ** P < 0.01 (one-way analysis of variance with Tukey’s post hoc test). (J) Brains lysates from APP/PS1ΔE9 mice were treated with phosphatase inhibitor (P.I.) or λ-protein phosphatase (λPP), and subjected to western blot to detect the indicated proteins. AD: Alzheimer’s disease; APP: amyloid precursor protein; DAPI: 4′,6-diamidino-2-phenylindole; DMSO: dimethyl sulfoxide; DNA: nuclear stained by DAPI that labels DNA; GSDMD: gasdermin D; GSDMD-FL: full-length GSDMD without being cleaved by active caspase-1; GSDMD-N: N-terminal fragment of GSDMD due to the cleavage of GSDMD-FL by active caspase-1; Iba1: ionized calcium binding adaptor molecule 1; NLRP3: NOD-, LRR- and pyrin domain-containing protein 3; ns: not significant; PCR: polymerase chain reaction; pro-caspase-1: The holo protein of caspase-1 without being cleaved; WT: wild-type.

Article Snippet: Subsequently, the membrane was incubated overnight at 4°C with gentle shaking with primary antibodies specific to the proteins of interest, including: anti-FUBP3 (rabbit polyclonal, 1:1000, PAG312Mu01, RRID: AB_3083686, Cloud-clone, Wuhan, Hubei, China), anti-NLRP3 (mouse monoclonal, 1:1000, AG-20B-0014-C100, RRID: AB_2885199, AdipoGen, San Diego, CA, USA), anti-caspase-1 (rabbit polyclonal, 1:1000, 2225S, RRID: AB_2243894, Cell Signaling, USA), anti-GSDMD (rabbit polyclonal, 1:1000, NBP2-33422, RRID: AB_2687913, NovusBio, MN, USA), anti-tau (rabbit polyclonal, 1:1000, PAV538Ge01, RRID: AB_3083687, Cloud-clone), anti-phosphorylated tau at site 181 (p-tau181; rabbit polyclonal, 1:1000, 28866-1-AP, RRID: AB_2881222, Proteintech, Wuhan, Hubei, China), anti-phosphorylated tau at sites 202/205 (p-tau202/205; mouse monoclonal, AT8, MN1020,1:1000, RRID: AB_223647, Invitrogen, Carlsbad, CA, USA), anti-PME1 (rabbit polyclonal, 1:1000, 14435-1-AP, RRID: AB_2284364, Proteintech), anti-phosphorylated-calmodulin (CaM)-dependent protein kinase IIα (CamKIIα; rabbit polyclonal, 1:1000, 12716, RRID: AB_2713889, Cell Signaling), anti-protein phosphatase 1 regulatory inhibitor subunit 1A (PPP1R1A; rabbit polyclonal, 1:1000, K108186P, RRID: AB_3083689, Solarbio, Beijing China), GAPDH (rabbit polyclonal, 1:1000, PAB932Hu01, RRID: AB_3083694, Cloud-clone), anti-β-actin (mouse monoclonal, 1:2000, PAB340Mi01, Cloud-clone, RRID: AB_3095306) or anti-histone H3 (rabbit polyclonal, 1:1000, PAA285Mi01, Cloud-clone, RRID: AB_3083688).

Techniques: Phospho-proteomics, Marker, Injection, Western Blot, Cell Culture, Mutagenesis, Transgenic Assay, Expressing, Staining, Reverse Transcription, Amplification, Negative Control, Binding Assay, Polymerase Chain Reaction