8052 nrf2 anti rabbit nrf2  (Cell Signaling Technology Inc)

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: RA-induced neurogenesis in P19 cells, assessed via qPCR analysis of ( a ) Oct4 and ( b ) Map2 mRNA levels, serving as pluripotent and neuronal markers, respectively. Additionally, ( c ) Nfe2l1 mRNA level was analysed to observe it’s expression changes during neurogenesis. Samples were collected at day 0 (WT cells), 6, and 10 post-cell plating and RA induction. Gapdh was used as the reference gene. Results are presented as fold change, with error bars indicating mean ± S.E.M. (n = 3 per group). Statistical significance denoted as ns (not significant), * p < 0.05, ** p < 0.01, and *** p < 0.001, among groups.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: Levels of Nfe2l1 protein in P19 cells. Western blotting confirmed the level of NFE2L1 protein in both P19 undiffrentiated cells and P19 neurons. Treatment with the proteasome inhibitor Mg132 (25 µM) resulted in ( a ) increased protein levels, as well as ( b , c ) upregulation of mRNA expression. * p < 0.05, ** p < 0.01, and *** p < 0.001 versus control.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Western Blot, Expressing, Control

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: NFE2L1 siRNA-mediated Knockdown. Transfection of ( a ) Undifferentiated and ( b ) P19 neurons with Nfe2l1 siRNA (si1 = siNEF2L1-1, si2 = siNEF2L1-2, and si3 = siNEF2L1-3) results in significant Knockdown of this gene. Error bars represent mean ± S.E.M. (n = 2 per group). Statistical significance is denoted as * p < 0.05, ** p < 0.01, *** p < 0.001, and ns (not statistically significant).

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Knockdown, Transfection

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: MAP2 expression is altered via NFE2L1 KD in P19 neurons. ( a ) Western blot analysis shows a significant reduction in protein levels of the neuronal marker MAP2 in NFE2L1 KD cells compared to siNC-treated control cells. ( b ) Quantification of western blotting shows a significant decrease in MAP2 protein levels in differentiated NFE2L1 siRNA-treated cells compared to siNC-treated control cells, with undifferentiated cells showing the lowest levels. Data are presented as mean ± SEM, n = 3 per group. Statistical significance was determined using one-way ANOVA with post hoc tests (*** p < 0.001, * p < 0.05). ( c ) qPCR results demonstrate a similar trend in MAP2 mRNA expression, with levels remaining significantly higher (** p < 0.01) in KD cells compared to undifferentiated cells, indicating a disruption in neurogenesis. Data are presented as mean ± SEM, (n = 3 per group). ( d ) Immunocytochemistry (ICC) confirms the presence of neuronal projections in control cells (siNC), while these connections are markedly absent in NFE2L1 KD cells. Although some signalling is detected in approximately half of the KD cells, the lack of established connections highlights the detrimental impact of NFE2L1 KD on neuronal differentiation and connectivity. Scale bar = 20 µm. ( e ) Quantification of MAP2 expression reveals a significant reduction in the corrected MAP2 intensity in NFE2L1 KD cells compared to control cells (*** p < 0.001, unpaired t-test, n = 6 per group). Data represent mean ± SEM.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Expressing, Western Blot, Marker, Control, Disruption, Immunocytochemistry

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: Further ubiquitination is triggered within NFE2L1 KD. In P19 neurons, western blot analysis ( a ) shows that poly-ubiquitinated proteins are present in both control groups, with higher levels observed in Mg132-treated cells compared to DMSO-treated cells. In the NFE2L1 KD group treated with Mg132, there is a more pronounced accumulation of ubiquitinated proteins compared to the Mg132-treated control group. ICC corroborates these findings, demonstrating increased levels of ubiquitinated proteins in ( c ) NFE2L1 KD cells relative to ( b ) control cells, scale bar = 20 µm. ( d ) Quantification of ubiquitin aggregates in ICC, reveals a significant increase in the number of aggregates in NFE2L1 KD cells compared to control cells (siNC). Data were analysed using One-way ANOVA with Tukey’s post-hoc test, and are presented as mean ± SEM. The number of ubiquitin aggregates per cell was counted for all 4 groups, based on 10 images taken from 2 biological replicates (5 images per replicate), with 100 cells counted per group. * p < 0.05, ** p < 0.01.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Ubiquitin Proteomics, Western Blot, Control

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: Impact of Nfe2l1 KD on PD-related gene expression in P19 neurons. qPCR analysis of P19 neurons shows that, among the 12 selected genes associated with PD and proteasome obtained from RNA sequencing, the Knockdown of Nfe2l1 significantly decreased mRNA levels of ( a ) Atf6 (** p < 0.01), ( b ) Camk2d (** p < 0.01), and ( c ) Sod1 (** p < 0.01) in the presence of Mg132. Data are mean ± SEM, n = 3 per group, one-way ANOVA with post hoc tests.

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Gene Expression, RNA Sequencing, Knockdown

Journal: Scientific Reports

Article Title: Nfe2l1 dysfunction alters Parkinson’s disease-related gene expression and impairs neuronal differentiation under ubiquitin stress in neuronal differentiated P19 Cells

doi: 10.1038/s41598-025-08204-x

Figure Lengend Snippet: Impact of Proteasome inhibition and Nfe2l1 KD on lncRNA expression. This Figure demonstrates the impact of proteasome inhibition on the expression of Malat1 and Neat1 lncRNAs, in undifferentiated and P19 neurons (neuronal diffrentiated). Our findings indicate a significant upregulation of both ( a ) Neat1 (*** p < 0.001) and ( b ) Malat1 (*** p < 0.001, ** p < 0.01) in Mg132-treated cells compared to control cells treated with DMSO. Additionally, Nfe2l1 KD resulted in a significant reduction in ( c ) Neat1 RNA levels (** p < 0.01), but no significant change was observed in ( d ) Malat1 RNA levels (ns). Error bars represent mean ± S.E.M. (n = 3 per group).

Article Snippet: The membranes were then blocked with 5% non-fat dry milk and incubated overnight at 4 °C with primary antibodies against NFE2L1 (Cat. #8052, Cell Signalling Technology, Danvers, MA, USA), MAP2 (Cat. #PA5-17,646, Thermo Fisher Scientific, Waltham, MA, USA, diluted 1:1000), ubiquitin (Cat. #sc-8017, Santa Cruz Biotechnology, Dallas, TX, USA, diluted 1:1000), OCT-4 (Cat. #2840, Cell Signaling Technology, Danvers, MA, USA, diluted 1:1000) and β-Actin (Cat. #4970, Cell Signalling Technology, Danvers, MA, USA) as a loading control.

Techniques: Inhibition, Expressing, Control