Journal: BioMed Research International

Article Title: Neuronal Nitric Oxide Synthase Is Dislocated in Type I Fibers of Myalgic Muscle but Can Recover with Physical Exercise Training

doi: 10.1155/2015/265278

Figure Lengend Snippet: Altered nNOS activity and protein expression. (a) Assessment of NOS activity in trapezius muscle from women with repetitive work tasks (MYA patients and CON) shows loss of activity in sarcolemma and gain of activity in sarcoplasm of large type I fibers compared to type II. Serial sections show NOS activity in black and MHC-I in red. # marks identical fibers on serial sections. Scale bar: 50 μ m. (b) Immunohistochemical staining of nNOS and MHC-II from trapezius and vastus lateralis muscle. In trapezius muscle type I fibers show reduced sarcolemma-localized nNOS protein and greater sarcoplasmic nNOS protein (∗) compared with type II fibers. Vastus lateralis did not show this type of alterations. nNOS in red, myosin heavy chain II in green, and a merged image with DAPI in blue. I and II mark identical type I and type II fibers, respectively. Scale bar: 50 μ m.

Article Snippet: Primary antibodies for myosin heavy chain II (MHC-II) (1 : 2000, M4276, Sigma Aldrich, Denmark), nNOS (1 : 200, AF2416, R&D Systems, United Kingdom), myosin heavy chain I (MHC-I) (1 : 2000, M8421, Sigma Aldrich, Denmark), α -dystroglycan (1 : 50, clone VIA4-1, Millipore, Germany), dystrophin (rod, 1 : 10, MAB1692, Millipore, Germany; C-terminal, 1 : 5, MAB1694, Millipore, Germany; N-terminal, 1 : 20, clone 34C5, Novocastra, United Kingdom), or laminin (polyclonal, Z0097, Dako, Denmark) were used.

Techniques: Activity Assay, Expressing, Immunohistochemical staining, Staining

Journal: BioMed Research International

Article Title: Neuronal Nitric Oxide Synthase Is Dislocated in Type I Fibers of Myalgic Muscle but Can Recover with Physical Exercise Training

doi: 10.1155/2015/265278

Figure Lengend Snippet: Intact dystrophin protein of type I fibers. Serial sections reveal normal dystrophin and laminin sarcolemmal protein expression in type I fibers displaying loss of NOS activity and reduced nNOS protein. NOS activity in black, dystrophin in white, laminin in blue, MHC-I in green, and nNOS in red. ∗ marks identical fibers on serial sections. Scale bar: 100 μ m.

Article Snippet: Primary antibodies for myosin heavy chain II (MHC-II) (1 : 2000, M4276, Sigma Aldrich, Denmark), nNOS (1 : 200, AF2416, R&D Systems, United Kingdom), myosin heavy chain I (MHC-I) (1 : 2000, M8421, Sigma Aldrich, Denmark), α -dystroglycan (1 : 50, clone VIA4-1, Millipore, Germany), dystrophin (rod, 1 : 10, MAB1692, Millipore, Germany; C-terminal, 1 : 5, MAB1694, Millipore, Germany; N-terminal, 1 : 20, clone 34C5, Novocastra, United Kingdom), or laminin (polyclonal, Z0097, Dako, Denmark) were used.

Techniques: Expressing, Activity Assay

Journal: BioMed Research International

Article Title: Neuronal Nitric Oxide Synthase Is Dislocated in Type I Fibers of Myalgic Muscle but Can Recover with Physical Exercise Training

doi: 10.1155/2015/265278

Figure Lengend Snippet: Intermyofibrillar network changes in muscle fibers lacking sarcolemmal nNOS. Enlarged type I fiber (#) with decreased sarcolemmal nNOS protein shows alterations in the intermyofibrillar network demonstrated by irregular NADH-TR staining ((a) and (b): arrow head). Note also a subsarcolemmal accumulation of cellular material or mitochondria seen as dark areas below the fiber membrane ((b): dark arrow). Type II fibers appear normal. NADH-TR (mitochondria) is blue, MHC-I is green, and nNOS is red. # marks identical fibers on serial sections. Scale bar: 50 μ m.

Article Snippet: Primary antibodies for myosin heavy chain II (MHC-II) (1 : 2000, M4276, Sigma Aldrich, Denmark), nNOS (1 : 200, AF2416, R&D Systems, United Kingdom), myosin heavy chain I (MHC-I) (1 : 2000, M8421, Sigma Aldrich, Denmark), α -dystroglycan (1 : 50, clone VIA4-1, Millipore, Germany), dystrophin (rod, 1 : 10, MAB1692, Millipore, Germany; C-terminal, 1 : 5, MAB1694, Millipore, Germany; N-terminal, 1 : 20, clone 34C5, Novocastra, United Kingdom), or laminin (polyclonal, Z0097, Dako, Denmark) were used.

Techniques: Staining, Membrane

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 2. Attenuation of mTOR prevents neurovascular uncoupling in 12-month-old hAPP(J20) mice. A, L-NPA at 200 nM has no impact on endothelium-dependent, eNOS-mediated, ACh- induced vasoreactivity. Pretreatment with L-NPA, a selective nNOS inhibitor, at 200 nM (close to its Ki for nNOS) has no impact on endothelium-dependent, eNOS-mediated vasoreactivity induced by superfusion of 10 mM ACh (Tukey’s test, q(8) = 0.94, p = 0.79 vs aCSF vehicle). In contrast, superfusion with 200 nM L-NPA together with 10 mM L-NAME, a general NOS inhibitor, profoundly inhibited ACh-induced CBF increases (91.7% inhibition, q(8) = 13.64, ppppp , 0.0001). B–D, NVC responses in hAPP mice; the fold change in CBF during whisker pad stimulation (30 s; bold black line) was measured sequentially in the presence of aCSF (vehicle; B), 200 nM L-NPA to inhibit nNOS (Fig. 2A; C), and 200 nM L-NPA plus 10 mM L-NAME (D) to inhibit all remain- ing NOS activity (i.e., eNOS). E, NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC is impaired in hAPP(J20) mice compared with WT littermates (q(27) = 10.41, ppppp , 0.0001). These deficits were negated by 8 months of rapamycin (Rapa; q(27) = 22.02, ppppp , 0.0001) and enhanced compared with WT (q(27) = 11.91, 1111p , 0.0001). Inhibition of nNOS with 200 nM L-NPA significantly reduced NVC in WT (q(27) = 5.24 vs baseline, pp = 0.02), but not in hAPP(J20) mice (q(27) = 0.38, p = 0.99 vs baseline), suggesting a preexisting nNOS deficit in hAPP(J20) animals that was negated by rapamycin treatment (q(27) = 7.14 vs baseline, pppp = 0.0008). The remaining NVC response in the presence of L-NPA plus L-NAME (inhibiting all remaining NOS activity; i.e., eNOS) was not significantly decreased with respect to L-NPA treatment in WT mice (q(27) = 2.31, p = 0.78) or in hAPP(J20) mice (q(27) = 0.76, p = 0.99) but was enhanced in rapamycin-treated hAPP(J20) mice (L-NPA1L-NAME vs

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Inhibition, Whisker Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 3. Short-term mTOR attenuation reverses NVC impairments in 12-month-old hAPP(J20) mice. A–C, Fold change in cerebral blood flow are the mean 6 SEM of n = 4–5/group during whisker pad stimulation (30 s, bold black line), stimulations conducted sequentially in the presence of aCSF [vehicle (Veh); A], 200 nM L-NPA to inhibit nNOS specifically (Fig. 2A; B), and 200 nM L-NPA110 mM L-NAME (C) to inhibit all NOS, thus defining the contribution of the non-nNOS-dependent, L-NAME-sensitive eNOS. D, NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC impairments in hAPP(J20) mice relative to WT (q(33) = 12.81, ppppp , 0.0001) are reversed by 2 months of rapamycin (Rapa; q(33) = 26.01, ppppp , 0.0001), with enhancement of NVC in rapamycin-treated hAPP(J20) mice compared with WT (q(33) = 13.93, ppppp , 0.0001). L-NPA superfusion significantly reduces NVC in WT mice (q(33) = 5.25, pp = 0.02), but not in hAPP(J20) mice (q(33) = 0.22, p . 0.99), suggesting a preexisting nNOS deficit in this group that is restored by rapamycin treatment in the hAPP(J20)1rapamycin group, indicated by a significant inhibition of NVC in the presence of 200 nM L-NPA (q(33) = 12.28 vs base- line, ppppp , 0.0001). The remaining NVC response in the presence of L-NPA1L-NAME (inhibiting all remaining NOS activity; i.e., eNOS) was not significantly different versus L-NPA alone in WT (q(33) = 1.05, p = 0.99), hAPP(J20) (q(33) = 0.09, p . 0.99), or in hAPP(J20)1rapamycin (q(33) = 3.60, p = 0.24). E, Contributions of nNOS, eNOS, and non-NOS components to total NVC. Deficits in nNOS-dependent NVC in hAPP(J20) mice (q(33) = 4.47, ppp = 0.009 vs WT) are negated by mTOR inhibition in rapamycin-treated hAPP(J20) mice (q(33) = 11.31, ppppp , 0.0001 vs hAPP) and nNOS-dependent NVC is enhanced over WT (q(33) = 7.10, 1111p , 0.0001). Additionally, rapamycin enhances L-NAME-sensitive activities (eNOS) in hAPP(J20) mice relative to vehicle-treated hAPP(J20) mice (q(33) = 3.93, pp = 0.02). Deficits in the remaining non-NO-mediated NVC response in hAPP(J20) mice (q(33) = 6.06, pppp = 0.0004 vs WT) are negated in rapamycin-treated hAPP(J20) mice [q(33) = 7.87, ppppp , 0.0001 vs vehicle-treated hAPP(J20) mice]. F, Hippocampal-dependent contextual memory impairment in 12-month-old hAPP(J20) mice [hAPP(J20) vs WT, q(16) = 4.19, pp = 0.024] is negated by 2 months of rapamycin treatment [q(16) = 3.93, pp = 0.034, hAPP(J20) vs hAPP(J20)1rapamycin]. G, Baseline NVC response is correlated with contextual memory performance (r = 0.668, p = 0.009). Behavioral studies used n = 6–7 mice/group. All post hoc analyses are Tukey’s multiple-comparisons tests. Unless other- wise indicated, asterisks (p) in the figure represent a significant difference relative to hAPP1Veh and pluses (1) represent a significant difference relative to WT1Veh.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Whisker Assay, Inhibition, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 4. mTOR-driven NVC deficits precede contextual memory impairment in 6-month-old hAPP(J20) mice. A, Hippocampal-dependent contextual memory is intact in 6-month-old hAPP (J20) mice (F(2,16) = 0.33, p = 0.73, no change). B–D, Fold change in cerebral blood flow from whisker pad stimulation (30 s, bold black line, representing mean 6 SEM of n = 5/group) was measured sequentially in the presence of aCSF [vehicle (Veh); B], 200 nM L-NPA to selectively inhibit nNOS (Fig. 2A; C), and 200 nM L-NPA110 mM L-NAME to inhibit all remaining NOS activity (i.e., eNOS; D). E, Total NVC responses during 30 s whisker stimulations. Area under the curve was calculated as an increase relative to baseline (i.e., only upward peaks). Baseline NVC impair- ment in 6-month-old hAPP(J20) mice versus WT (q(35) = 8.71, 1111p , 0.0001) is reversed by rapamycin (Rapa) treatment (2months, q(35) = 21.07, ppppp , 0.0001) and NVC is enhanced in hAPP(J20)1rapamycin compared with WT (q(35) = 12.36, 1111p , 0.0001). A significant reduction in NVC by L-NPA superfusion (q(35) = 11.75, ppppp , 0.0001) in the hAPP(J20)1rapamycin group, which is absent in vehicle-treated hAPP(J20) mice (q(35) = 0.26, p . 0.99), indicates that rapamycin restores nNOS-dependent NVC in hAPP(J20) mice. L- NPA1L-NAME-sensitive (i.e., eNOS-mediated) NVC was not significantly reduced in WT (q(35) = 3.40, p = 0.32), hAPP(J20) (q(35) = 0.87, p = 0.99), or hAPP(J20)1rapamycin groups (q(35) = 3.04, p = 0.46) relative to L-NPA alone. F, Contributions of nNOS, eNOS, and non-NOS components to total NVC. Impaired L-NPA-sensitive, nNOS-driven NVC responses in hAPP(J20) mice (q(35) = 3.67, pp = 0.036) are negated by rapamycin (2months, q(35) = 11.28, ppppp , 0.0001), and the nNOS-driven NVC response is enhanced versus WT groups (q(35) = 7.60, 1111p , 0.0001). eNOS-mediated, L-NPA1L-NAME-sensitive NVC is unchanged at 6 months of age in all groups; mTOR attenuation enhances non-NOS-dependent NVC in rapamycin-treated hAPP(J20) mice [q(35) = 4.27, 1p = 0.013 vs WT mice; q(35) = 7.09, ppppp , 0.0001 vs hAPP(J20) mice]. G, Progressive, early-onset NVC impairments in hAPP(J20) mice modeling AD. NVC impairments in hAPP(J20) mice are present at 6 months (q(23) = 6.18, 11p = 0.003) and are significantly exacerbated by 12 months of age (q(23) = 5.55, ppp = 0.008). During this same time period, NVC remains unchanged in both WT animals (q(23) = 1.17, p = 0.96, not significant) and in hAPP(J20) mice treated with rapamycin (q(23) = 2.10, p = 0.67, not significant). All post hoc tests are Tukey’s multiple-comparisons tests. N = 5 mice/group except for behavioral studies (n = 6–7/group). Unless otherwise indicated, asterisks (p) in the figure represent a significant dif- ference relative to hAPP1Veh and pluses (1) represent a significant difference relative to WT1Veh.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Whisker Assay, Activity Assay

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 5. mTOR decreases nNOS abundance and inhibits nNOS phosphorylation at its primary activation site in cultured neuronal cells. A, Representative electropherograms of chemilumines- cent signals detected by capillary Western immunoassay (Wes; Beekman et al., 2018) measuring total nNOS from hippocampal formation of patients at Braak stages III–VI compared with non- AD controls (C). B, Quantitative analysis shows decreased nNOS abundance in the hippocampal formation with progression of Braak pathology (Spearman’s r = 0.975, p = 0.02). Data are a scatter plot overlaid with the regression line and 95% CI representing n = 3/group. C, Representative electropherograms of chemiluminescent signals detected by capillary Wes measuring total nNOS (152 kDa), eNOS (140 kDa), and b -actin (48 kDa) in isolated microvasculature from hAPP(J20) mice at 6 or 12months (mo.) of age that received 2 months of vehicle (Veh) or rapamycin (Rapa)-containing diets. D, Total nNOS is reduced in 6-month-old hAPP(J20) mice modeling early-stage preclinical AD (Sidak’s test, t(17) = 5.42, p = 0.0001). Two months of mTOR attenuation restored nNOS expression in hAPP(J20) mice treated with rapamycin (Sidak’s test, t(17) = 7.37, p , 0.0001). No change in total nNOS was observed in 12-month-old hAPP(J20) mice. E, Total eNOS protein expression is unchanged by age (F(2,18) = 0.005, p = 0.94), treatment (F(2,18) = 1.19, p = 0.33), or their interaction (F(2,18) = 2.67, p = 0.10). F, Representative electropherograms of chemiluminescent signals detected by capillary Wes in lysates from N2a cells. G–J, Quantitative analyses of data in F indicate that rapamycin (5.5 nM) treatment inhibits mTOR in N2a cells (81.7% reduction in S6 phosphorylation, t(10) = 23.5, ppppp , 0.0001; G); increases nNOS Ser1412 phosphorylation (t(10) = 31.2, ppppp , 0.0001; H) and total nNOS levels (t(14) = 9.3, ppppp , 0.0001; I); and increases Hsp90 levels (t(10) = 4.0, ppp = 0.003) in N2a cells (J). Data are scatter plots for n = 8 controls and n = 4 rapamycin-treated N2a cultures, from n = 4 inde- pendent experiments performed on different days, overlaid on a bar graph representing mean 6 SEM.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Phospho-proteomics, Activation Assay, Cell Culture, Western Blot, Isolation, Expressing

Journal: The Journal of Neuroscience

Article Title: mTOR Attenuation with Rapamycin Reverses Neurovascular Uncoupling and Memory Deficits in Mice Modeling Alzheimer's Disease

doi: 10.1523/jneurosci.2144-20.2021

Figure Lengend Snippet: Figure 7. mTOR-dependent regulation of cerebrovascular function through nitric oxide synthases. mTOR inhibits neurovascular coupling through the regulation of nNOS-, eNOS-, and non- NO-dependent components of the neurovascular coupling response. Our prior studies indicate that mTOR drives cerebrovascular reactivity deficits and reduced baseline cerebral blood flow through the inhibition of eNOS activity (Van Skike and Galvan, 2018) in models of AD ( Lin et al., 2013, 2017; Van Skike et al., 2018), in models of vascular cognitive impairment (Jahrling et al., 2018; Van Skike et al., 2018), and in normative aging (Van Skike et al., 2020). Figure created in part with BioRender.

Article Snippet: Primary antibodies for ribosomal protein S6 (rpS6; catalog #2217, CST), phospho-rpS6 (S240/S244; catalog #2215, CST), nNOS (catalog #sc-5302, Santa Cruz Biotechnology), phospho-nNOS (S1412; catalog #NBP1-97488, Novus Biologicals), eNOS (catalog #AF950, R&D Systems), Hsp90 (catalog #4877, CST), vinculin (catalog #MCA465GA, BIO-RAD), and b -actin (catalog #4970, CST) were used with the appropriate anti-mouse, anti-rabbit, or anti-goat secondary antibodies (ProteinSimple).

Techniques: Inhibition, Activity Assay

Journal: Brain research bulletin

Article Title: Hindbrain Dorsal Vagal Complex AMPK Controls Hypothalamic Gluco-regulatory Transmitter and Counter-Regulatory Hormone Responses to Hypoglycemia

doi: 10.1016/j.brainresbull.2018.11.016

Figure Lengend Snippet: Impact of Cc Pretreatment on Ventromedial Hypothalamus Nucleus (VMN) AMPK and, Glutamate Decarboxylate65/67 (GAD65/67), and Neuronal Nitric Oxide Synthase (nNOS) Protein Expression during Insulin-Induced Hypoglycemia (IIH). Results show mean normalized VMN AMPK (Panel A), pAMPK (Panel B), GAD65/67 (Panel C), and nNOS (Panel D) O.D. values + S.E.M. for V/V, V/INS, and Cc/INS treatment groups (n=5/group). *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: Proteins of interest were probed with primary polyclonal antisera raised in rabbit against AMPK α1/2 (1:2,000; 2532s; Cell Signaling Technology, Danvers, MA), pAMPK α1/2 (Thr 172; 1:2,000; 2535; Cell Signaling Technol.), RFamide related peptide-1 (RFRP-1; 1:1,000, sc-67010; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), or neuronal nitric oxide synthase (nNOS; 1:2,000, NBP1–39681; Novus Biologicals, LLC, Littleton, CA), or raised in goat against NPY (1:2,500, NBP1–46535; Novus Biol.), POMC (1:2,000, NB100–1533; Novus Biol.), GAD 65/67 (1:2,000, AB1511; EMD Millipore, Burlington, MA), RFamide related peptide-3 (RFRP-3; 1:1,000, sc-32380; Santa Cruz Biotechnol.), or ORX-A (1:1,000, sc-8070; Santa Cruz Biotechnol.).

Techniques: Expressing

Journal: Dermatology Practical & Conceptual

Article Title: GRWD1 Drives Melanoma Growth Through NF-κB Signaling Pathway

doi: 10.5826/dpc.1504a5918

Figure Lengend Snippet: GRWD1 knockdown suppresses invasion and oncogenic signaling in melanoma cells. (A) Matrigel invasion assay shows significantly reduced invasion capacity in GRWD1 siRNA-treated melanoma cells (**** P <0.001). (B) Western blot analysis shows reduced Bcl-2 expression and increased cleaved PARP levels in GRWD1 knockdown cells, indicating apoptosis. (C) Western blot analysis of key oncogenic signaling proteins. GRWD1 knockdown leads to decreased NF-κB, Src, and MDM2 expression while increasing p53 levels. (D) Validation of GRWD1 knockdown effects in A2780 ovarian cancer cells. Cell viability is significantly reduced after GRWD1 knockdown (* P <0.05, * P <0.01). Flow cytometry and Western blot data represent mean ± SD from three independent experiments. Statistical comparisons were made using one-way ANOVA (Tukey’s post hoc test).

Article Snippet: Membranes were incubated overnight at 4° C with primary antibodies against GRWD1 (Biolegend, Cat. No: 645702), Bcl-2 (Elabscience, Cat. No: E-AB-22004), cleaved PARP (Cell Signaling, Cat. No: D214, 19F4), P53 (Biolegend, Cat. No: 645702), p21 (BT LAB, Cat. No: BT-AP12602), MDM2 (Biogend, Cat. No: E-AB-60076), NF-κB p65 (St John’s Laboratory, Cat. No: STJ903553-100), Src (Cell Signaling, Cat. No: 2108S), and β-Actin (Santa Cruz, Cat. No: sc-47778) as a loading control.

Techniques: Knockdown, Invasion Assay, Western Blot, Expressing, Biomarker Discovery, Flow Cytometry

Journal: Dermatology Practical & Conceptual

Article Title: GRWD1 Drives Melanoma Growth Through NF-κB Signaling Pathway

doi: 10.5826/dpc.1504a5918

Figure Lengend Snippet: GRWD1 promotes the ability of proliferation, survival and invasion in melanoma cells through the activation of NF-κB pathway. Proposed mechanism underlying GRWD1-mediated melanoma progression. GRWD1 enhances NF-κB signaling, which transcriptionally upregulates Bcl-2 and activates Src. NF-κB also induces MDM2 expression, promoting p53 degradation. The downregulation of p53 relieves transcriptional repression of Bcl-2, further supporting cell survival and proliferation. This GRWD1–NF-κB–MDM2/p53–Bcl-2 axis contributes to melanoma pathogenesis. Arrows represent activation (solid) and inhibition (dashed).

Article Snippet: Membranes were incubated overnight at 4° C with primary antibodies against GRWD1 (Biolegend, Cat. No: 645702), Bcl-2 (Elabscience, Cat. No: E-AB-22004), cleaved PARP (Cell Signaling, Cat. No: D214, 19F4), P53 (Biolegend, Cat. No: 645702), p21 (BT LAB, Cat. No: BT-AP12602), MDM2 (Biogend, Cat. No: E-AB-60076), NF-κB p65 (St John’s Laboratory, Cat. No: STJ903553-100), Src (Cell Signaling, Cat. No: 2108S), and β-Actin (Santa Cruz, Cat. No: sc-47778) as a loading control.

Techniques: Activation Assay, Expressing, Inhibition