Journal: Evidence-based Complementary and Alternative Medicine : eCAM

Article Title: Effects of Combined Treatment with Acupuncture and Chunggan Formula in a Mouse Model of Parkinson's Disease

doi: 10.1155/2019/3612587

Figure Lengend Snippet: Protective effects of the combined treatment in Parkinson's disease mouse model. The SNpc was isolated and immunoblotted with the indicated antibodies. The intensities of the protein bands were quantitated by densitometry, and the phosphorylated forms were normalized to the total form or β -actin. Changes in (a) pI κ B α , (b) pAKT, (c) pGSK3 β , (d) pERK, (e) pCREB, and (f) BDNF. Data are mean ± standard error. ∗∗∗ p < 0.001 by one-way ANOVA followed by the Newman–Keuls post hoc test ( n = 4 for each group).

Article Snippet: The membranes were washed in tris-buffered saline with 0.1% Tween 20 (TBS-T) three times for 10 min and then blocked in 5% skim milk for 1 h. After blocking, the membranes were activated with antibodies to brain-derived neurotrophic factor (BDNF, 1 : 200, Santa Cruz Biotechnology; sc-546), TH (1 : 3000, Santa Cruz Biotechnology, sc-14007), β -actin (1 : 40,000, Sigma-Aldrich, St. Louis, MO, USA; A1978), phospho-inhibitory kappa B alpha (pI κ B α Ser32, 1 : 500, Cell Signaling Technology, Beverly, MA, USA; #2859), I κ B α (1 : 500, Cell Signaling Technology, #4814), phospho-extracellular signal-regulated kinase (pERK Thr202/Tyr204, 1 : 500, Cell Signaling Technology; #4370), ERK (1 : 500, Cell Signaling Technology, #9102), phospho-protein kinase B (pAKT Ser473, 1 : 1000, Cell Signaling Technology, #4058), AKT (1 : 1000, Cell Signaling Technology, #4691), phospho-cAMP response element-binding protein (pCREB Ser133, 1 : 200, Cell Signaling Technology, #9198), CREB (1 : 500, Cell Signaling Technology, #9197), phospho-glycogen synthase kinase 3 beta (pGSK3 β Ser9, 1 : 250, Cell Signaling Technology, #9336), and GSK3 β (1 : 200, Cell Signaling Technology, #9315) overnight at 4°C.

Techniques: Isolation

Journal: PLoS ONE

Article Title: Microtubule Destabilization Is Shared by Genetic and Idiopathic Parkinson’s Disease Patient Fibroblasts

doi: 10.1371/journal.pone.0037467

Figure Lengend Snippet: (A) Immunoblot and densitometric analyses of (B) total and (C) phosphorylated glycogen synthase kinase 3 beta (GSK3β), p38 MAP Kinase (p38) and p44/42 MAPK (Erk) were performed in whole cell extracts from human fibroblasts deriving from control (CONT, white bars, N = 3), mutated parkin (PARK, dark grey bars, N = 3), mutated LRRK2 (LRRK2, light grey bars, N = 3) and idiopathic PD (PD, black bars, N = 3). For the quantitation, values of total protein were normalized on the level of GAPDH of the relative sample, whereas the levels of phosphorylated form were normalized on the values of total protein. All values are expressed as mean ± SEM. * p <0.05 and ** p <0.02 vs control, # p <0.05 vs PD according to ANOVA, Tukey HSD post hoc test.

Article Snippet: Membranes were probed with the following antibodies: α-tubulin mouse IgG (clone B-5-1-2, Sigma-Aldrich, St Louis, MO); β-tubulin mouse IgG (clone Tub 2.1, Sigma-Aldrich); deTyr tubulin rabbit IgG (Chemicon, Temecula, CA); Tyr tubulin mouse IgG (clone TUB-1A2, Sigma-Aldrich); Ac tubulin mouse IgG (clone 6-11B-1, Sigma-Aldrich); microtubule-associated protein 1 light chain 3 rabbit IgG (Sigma-Aldrich); vimentin mouse IgG (clone V6, Sigma-Aldrich); actin mouse IgM (N350, Amersham, Little Chalfont, UK); Caspase 3 rabbit IgG (Enzo Life Sciences Ag., Lausen, Switzerland), GADPH mouse IgG (Biogenesis, Poole, UK); Heat Shock Protein 70 mouse IgG (clone 3A3, Chemicon); Glycogen synthase kinase 3 beta rabbit IgG (Abcam, Cambride, UK); Phospho-Glycogen synthase kinase 3 beta (Ser9) rabbit IgG (Cell Signaling Technology, Beverly, MA); p38 alpha MAP Kinase mouse IgG (clone L53F8, Cell Signaling Technology); Phospho-p38 MAP Kinase (Thr180/Tyr182) rabbit IgG (clone 3D7, Cell Signaling Technology); p44/42 MAPK (Erk1/2) rabbit IgG (clone 137F5, Cell Signaling Technology); Phospho-p44/42 MAPK (Thr202/Tyr204) rabbit IgG (clone D13.14.4E, Cell Signaling Technology); parkin mouse IgG (clone prk8, Sigma-Aldrich).

Techniques: Western Blot, Quantitation Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: BGP-15 Protects against Heart Failure by Enhanced Mitochondrial Biogenesis and Decreased Fibrotic Remodelling in Spontaneously Hypertensive Rats

doi: 10.1155/2021/1250858

Figure Lengend Snippet: Effect of BGP-15 treatment on the phosphorylation of Akt-1 Ser473 and GSK-3 β Ser9 . Representative Western blot analysis of Akt-1 and GSK-3 β phosphorylation and densitometric evaluation are shown. GAPDH was used as a loading control. WKY: age-matched normotensive Wistar-Kyoto rats, n = 7; SHR-Baseline: 15-month-old spontaneously hypertensive rats, n = 7; SHR-C: nontreated spontaneously hypertensive rats, n = 7; SHR-B: spontaneously hypertensive rats receiving BGP-15 for 18 weeks, n = 7. Values are mean ± SEM. ∗∗ p < 0.01 vs. WKY, ## p < 0.01 vs. SHR-Baseline, § p < 0.05 vs. SHR-C, §§ p < 0.01 vs. SHR-C.

Article Snippet: After blocking (2 h with 5% BSA in Tris-buffered saline contained with 1% Tween-20), membranes were probed overnight at 4°C with primary antibodies recognizing the following antigens: transforming growth factor- β (TGF- β ; 1 : 1000; Cell Signaling #3711), Smad2 (1 : 1000; Invitrogen, 436500), phospho-specific Smad2 Ser465/467 (1 : 1000; Invitrogen, MA5-15122), Smad3 (1 : 1000; Cell Signaling #9523), phospho-specific Smad3 Ser423/425 (1 : 1000; Cell Signaling #9520), protein kinase B (Akt; 1 : 1000; Cell Signaling #9272), phospho-specific Akt-1/protein kinase B- α Ser473 (1 : 1000; Cell Signaling #4060), glycogen synthase kinase-3 β (GSK-3 β ; 1 : 1000; Cell Signaling #9832), phospho-specific glycogen synthase kinase-3 β Ser9 (1 : 1000; Cell Signaling #5558), p38 mitogen-activated protein kinase (p38MAPK; 1 : 1000; Cell Signaling #8690), phospho-specific p38 mitogen-activated protein kinase Thr180/Tyr182 (1 : 1000; Cell Signaling #4511), c-Jun N-terminal kinase (JNK; 1 : 1000; Cell Signaling #9252), phospho-specific c-Jun N-terminal kinase Thr183/Tyr185 (1 : 1000; Cell Signaling #9255), extracellular signal-regulated kinase (ERK1/2; 1 : 1000; Cell Signaling #4695), phospho-specific extracellular signal-regulated kinase 1/2 Thr202 (1 : 1000; Cell Signaling #4370), mitogen-activated protein kinase phosphatase-1 (MKP-1; 1 : 100; Santa Cruz Biotechnology, sc-373841), peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1 α ; 1 : 1000; Novus Biologicals, NBP1-04676), cAMP response element-binding protein (CREB; 1 : 1000; Cell Signaling #4820), phospho-specific cAMP response element-binding protein Ser133(1 : 1000; Cell Signaling #9198), 5′ AMP-activated protein kinase (AMPK; 1 : 1000; Cell Signaling #2532), phospho-specific 5′ AMP-activated protein kinase Thr172 (1 : 1000; Cell Signaling #2535), and voltage-dependent anion channel (VDAC; 1 : 1000; Cell Signaling #4661).

Techniques: Western Blot

Journal: Cells

Article Title: Downregulation of LKB1/AMPK Signaling in Blood Mononuclear Cells Is Associated with the Severity of Guillain–Barre Syndrome

doi: 10.3390/cells11182897

Figure Lengend Snippet: AMPK–ERK–PKC interplay in GBS PBMCs. ( a , b ) PBMCs were isolated from control subjects (CTRL; n = 20) and GBS patients ( n = 23), and the levels of phosphorylated ERK, p38 MAPK, and GSK3β were assessed by immunoblotting. The representative blots and densitometry data (relative to actin) are shown in ( a ), with horizontal lines representing median values and interquartile range (* p < 0.05, two-tailed Mann–Whitney U test). The correlations between GSK3β and phospho-AMPK, phospho-LKB1, or phospho-ERK are presented in ( b ) (r s —Spearman’s correlation coefficient; * p < 0.05, Spearman’s rank-order test).

Article Snippet: After blocking with 10% milk powder, membranes were incubated with primary rabbit antibodies against SQSTM1 (NBP1-48320; Novus Biologicals, Littleton, CO, USA), NBR1 (#9891), LC3B (#2775), beclin-1 (#3495), ATG5 (#12994), LKB1 (#3047), phospho-LKB1 (Ser428; #3482), AMPKα (#2603), phospho-AMPKα (Thr172; #2535), Raptor (#2280), phospho-Raptor (Ser792; #2083), phospho-eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) (Thr37/46; #2855), phospho-AKT (Ser473; #9271), phospho-ERK1/2 (Thr202/Tyr204; #9101), phospho-p38 MAPK (Thr180/Tyr182; #9211), phospho-glycogen synthase kinase 3β (GSK3β, Ser9; #9322), and actin (#4968) as a loading control (all from Cell Signaling Technology, Cambridge, MA, USA).

Techniques: Isolation, Western Blot, Two Tailed Test, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: Endoplasmic reticulum stress downregulates PGC-1α in skeletal muscle through ATF4 and an mTOR-mediated reduction of CRTC2

doi: 10.1186/s12964-022-00865-9

Figure Lengend Snippet: mTORC1 inhibition prevents the reductions in PGC-1α, IRS1 and the insulin signaling pathway caused by ER stress. A mRNA levels and B , C protein levels in human LHCN-M2 myotubes incubated with 250 nM torin1 or 100 nM rapamycin and/or 5 µg/ml of tunicamycin for 16 h (for PGC-1α, IRS1, total GSK3 α/β and phospho-GSK3 α/β (Ser21/9)) or 2 h (for total Akt and phospho-Akt (Ser 473)). D LHCN-M2 myotubes incubated with 100 nM rapamycin and/or 5 µg/ml of tunicamycin for 16 h with or without 100 nM of insulin (Ins) for the last 30 min. Data are presented as the mean ± SEM (n = 3–5 per group). * p < 0.05, ** p < 0.01 and *** p < 0.001 versus control. # p < 0.05, ## p < 0.01 and ### p < 0.001 versus tunicamycin. && p < 0.01 versus insulin-stimulated cells

Article Snippet: Immunoblotting was performed with antibodies against PGC-1α (Abcam, Cambridge, UK), α-actinin, histone H2B, CRTC2, ATF4, insulin receptor substrate 1 (IRS1), TRIB3 (Santa Cruz Biotechnology Inc.), and tumour necrosis factor (TNF)-α, total and phospho-S6 ribosomal protein (Ser235/236), insulin receptor β, GAPDH, p44/42 MAPK(ERK1/2), phospho-p44/42 MAPK (Thr202/Tyr204) or phospho-CREB (Ser133), total and phospho-glycogen synthase kinase (GSK)3-α/β (Ser21/9), total and phospho-Akt (Ser473) (Cell Signaling, Danvers, MA), and CRTC2 (Calbiochem, San Diego, CA).

Techniques: Inhibition, Incubation

Journal: Annals of Translational Medicine

Article Title: LncRNA MALAT1 induced by hyperglycemia promotes microvascular endothelial cell apoptosis through activation of the miR-7641/TPR axis to exacerbate neurologic damage caused by cerebral small vessel disease

doi: 10.21037/atm-21-5997

Figure Lengend Snippet: Regulation of TPR expression affects the phosphorylation activity of the Akt pathway. (A) The expression level of TPR protein in each group detected by WB to verify the transfection effect; (B) the expression level of p-Akt/Akt, and p-GSK3β/GSK3β proteins in each group detected by WB; (C) lncRNA MATAL1/miR-7641/TPR pathway diagram. **P<0.01, ***P<0.001. WB, Western blot; TPR, translocated promoter region; lncRNA, long noncoding RNA; MALAT1, metastasis-associated lung carcinoma transcript 1; Nor, normal; Nor + TPR inhibitor, normal + TPR inhibitor; HG, high glucose; TPR oe, TPR over express; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; p-GSK3β, phospho-glycogen synthase kinase-3β.

Article Snippet: The antibodies included anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody [6C5]-Loading Control (ab8245) (1:1,000), anti-von Willebrand factor (vWF) antibody [ {"type":"entrez-protein","attrs":{"text":"EPR12011","term_id":"523377596","term_text":"EPR12011"}} EPR12011 ] (ab174290) (1:2,000), anti-intercellular adhesion molecule-1 (ICAM1) antibody [ {"type":"entrez-protein","attrs":{"text":"EPR16608","term_id":"523382629","term_text":"EPR16608"}} EPR16608 ] (ab179707) (1:1,000), anti-Bcl-2 antibody (ab196495) (1:1,000), anti-caspase-3 antibody [31A1067] (ab13585) (1:50), anti-Akt antibody (ab8805) (1:500), anti-Akt (phospho T308) antibody (ab38449) (1:1,000), anti-GSK3 beta antibody (ab93926) (1:1,000), and phosphorylated glycogen synthase kinase 3 beta (p-GSK3β) (Ser9) (1:1,000, cat no. 9336, CST).

Techniques: Expressing, Activity Assay, Transfection, Western Blot