Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 2. Inhibition of ULK1 results in a decrease of pICln phosphorylation in vivo A, Colocalization of ULK1 and pICln phospho and total. Cells were fixed with 4% PFA and afterwards permeabilized with Triton X-100 to visualize ULK1 (red) and pICln total or phosphospecific pICln (green). The DNA was stained with DAPI (blue), scale bars 10 µm. B, HEK293T cells were treated with 30 µM ULK inhibitor MRT67307 for 5 h or transfected with 50 nM Non-targeting control pool (NT) control or ULK1 siRNA for 72 h. Cells were fixed as described in A to visualize ULK1 (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. C, HEK293T cells were treated as described in B to visualize phosphospecific pICln (red) and pICln total (green). The DNA was stained with DAPI (blue), scale bars 10 µm. D, Inhibition or knockdown of ULK1 decreases pICln phosphorylation in vivo. Quantification of the intensity ratio between phosphospecific pICln and pICln total was made in Fiji and the diagram in Origin, the values for MRT67307 treatment are normalized on HEK wt value, and the values for ULK1 siRNA are normalized on NT control. The significance of the values was analyzed using a Mann-Whitney U test, the p-value of HEK wt vs. MRT67307 and NT control vs. ULK1 siRNA was below 0.005, indicating significant differences between the values.

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Inhibition, Phospho-proteomics, In Vivo, Staining, Transfection, Control, Knockdown, MANN-WHITNEY

Journal: Computational and structural biotechnology journal

Article Title: Phosphorylation of pICln by the autophagy activating kinase ULK1 regulates snRNP biogenesis and splice activity of the cell.

doi: 10.1016/j.csbj.2023.03.015

Figure Lengend Snippet: Fig. 4. ULK1 phosphorylation of pICln increases UsnRNP biogenesis and spliceosomal activity A, The HIV-1-based splicing reporter contains a test 5‘splice site (5‘ss) with a mutated version of viral 5′ss D4 termed „− 1G3U“. This splice site carries two nucleotide substitutions: A-to-G at position − 1 (−1 G) and A-to-U at position + 3 (+3 U) and is poorly recognized by the endogenous U1 snRNA due to a mismatch at position + 3. However, splice site recognition is efficiently restored following the co-expression of a U1 snRNA with a compensatory U-to-A nucleotide exchange at position + 6, indicating that the modified U1 is assembled into functional snRNP particles within the cell. Uppercase letters within the splice site sequences represent complementary residues, while lowercase letters represent mismatches to the U1. Mutations are highlighted in red. Base-pairing at position + 3 is highlighted by a light red box and increased font size. B; C, HEK293T cells were treated with 30 µM inhibitor MRT67307 and transiently transfected with different splicing reporter constructs (see methods section for more details). After 20 h of inhibitor treatment cells were harvested to perform RNA isolation. Quantitative RT-PCR was executed and mRNA was analyzed by assessing the respective ratio of spliced/unspliced form, the p value was below 0.005 (B). The equal averages including differences between inhibitor- treated and untreated spliced and unspliced forms are listed in C (n = 3).

Article Snippet: To control whether ULK1 siRNA worked and if the inhibition or knockdown of ULK1 total has an effect on pICln total localization the following antibodies were used: ULK1 Prestige Antibody Sigma (HPA063990) rabbit host and pICln C5 antibody Santa Cruz mouse host (sc-130668).

Techniques: Phospho-proteomics, Activity Assay, Expressing, Modification, Functional Assay, Transfection, Construct, Isolation, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Autophagy proteins regulate ERK phosphorylation

doi: 10.1038/ncomms3799

Figure Lengend Snippet: ( a ) EGF enhances colocalization of phosphorylated (P)-bRAF, P-MEK and P-ERK with LC3. Immunofluorescence (IF) showing colocalization of P-bRAF (green), P-MEK (green) and P-ERK (green) with LC3 (red) in NIH/3T3 cells in presence or absence of EGF (10 min as shown in scheme). The bars represent mean±s.e.m. ** P <0.01, *** P <0.001; Student’s t -test, 60 cells analysed from two experiments. Scale bars, 10 μm. ( b ) P-ERK colocalizes with pre-autophagosomal and autophagosomal structures. IF depicting colocalization of P-ERK (green or red) with LC3 (red), ATG5–ATG12 (red), ATG16 (green), vps34 (red), WIPI1 (red), WIPI2 (red) and P-ULK1 (red) in EGF-treated NIH/3T3 cells. Scale bars, 10 μm. The bars represent mean±s.e.m. 50 cells analysed from two experiments. ( c ) Growth factors in serum maintain P-ERK/LC3 colocalization. IF depicting colocalization of P-ERK (green) with LC3 (red) in 2 h serum-fed NIH/3T3 cells. ( a – c ) Native merged images or images with colocalization highlighted in white pixels are shown. Nuclei are blue (DAPI). Scale bar, 10 μm.

Article Snippet: Antibodies ( ) for ATG7, ATG4B, LC3B, P-bRAF (Ser445), bRAF, P-cRAF (Ser338), cRAF, P-p90RSK (Thr359/Ser363), P-MEK1/2 (Ser217/221), MEK1/2, P-ERK1/2 (Thr202/Tyr204) (rabbit and mouse species-specific), ERK1/2, ERK1, ERK2, P-Elk1 (Ser383), P-mTOR (Ser2448), mTOR and P-ULK1 (Ser555) were from Cell Signaling (Danvers, MA, USA); ATG5–ATG12 and ULK1 from Novus Biologicals (Littleton, CO, USA); p62 from Enzo life sciences (Plymouth Meeting, PA, USA); ATG16, Cathepsin B, Cathepsin L, GABARAP and GATE16 (sc-28938), P-c-jun (Ser63/73), c-jun, P-JNK (Thr183/Tyr185), JNK, IκBα, Rab7 and TFEB were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Beclin1 from BD Biosciences (San Jose, CA, USA); GAPDH, WIPI1 and WIPI2 antibodies from Abcam (Cambridge, MA, USA); Histone3 from Millipore (Billerica, MA, USA), β-actin from Sigma-Aldrich and JL-8 for CFP detection from Clontech (Mountain View, CA, USA).

Techniques: Immunofluorescence

Journal: Frontiers in Cell and Developmental Biology

Article Title: The effect of 4.3 GHz high-power microwave exposure on human corneal epithelial cells

doi: 10.3389/fcell.2026.1729198

Figure Lengend Snippet: Protein-level validation of the AMPK–TSC2–mTOR–ULK1 signaling pathway at 24 h after 4.3 GHz HPM exposure. Representative Western blot bands and quantification of total and phosphorylated signaling proteins, including AMPK, phosphorylated AMPK (p-AMPK) (A–C) TSC2, and phosphorylated TSC2 (p-TSC2) (D–F) mTOR, phosphorylated mTOR (p-mTOR) (G–I) ULK1, phosphorylated ULK1 (p-ULK1) (J–L) in HCE-T cells following exposure to 1.64 W/kg, 3.28 W/kg, and 8.2 W/kg HPM for 24 h. Protein expression levels were normalized to the corresponding total protein (for phosphorylated forms) or β-actin (for total protein). (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: ULK1 (1:500, 20986-1-AP), p-ULK1 (Ser555) (1:500, 80218-1-RR), TSC2 (1:1000, 24601-1-AP), p-TSC2 (1:500, 29000-1-AP), AMPK (1:5000, 10929-2-AP) were from Proteintech, Wuhan, China.β-actin (1:5000, GB15003-100), HRP conjugated Goat Anti-Rabbit IgG (H + L) (1:3000, GB23303) were from Servicebio, Wuhan, China.

Techniques: Biomarker Discovery, Western Blot, Expressing

Journal: Frontiers in Pharmacology

Article Title: Escins Isolated from Aesculus chinensis Bge. Promote the Autophagic Degradation of Mutant Huntingtin and Inhibit its Induced Apoptosis in HT22 cells

doi: 10.3389/fphar.2020.00116

Figure Lengend Snippet: EB regulates both the mTOR and ERK signaling pathways. HT22 cells were treated with EB at the indicated concentrations for 24 h. Cell proteins were then harvested for the Western blot analysis of p-PI3K, PI3K, p-Akt, Akt, p-AMPK, AMPK, p-mTOR, mTOR, p-ULK1, ULK1, p-P70S6K, P70S6K, and β-actin (A) , and p-Raf, Raf, p-MEK, MEK, p-ERK, ERK, and β-actin (B) . Bar chart indicates the relative expression of phosphorylation protein to total protein; bars, S.D. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001. The full-length Western blot images are showed in .

Article Snippet: Huntingtin (sc-47757) and ULK1 (sc-390904) came from Santa Cruz Biotechnology Co., Ltd. (Dallas, Texas, USA).

Techniques: Protein-Protein interactions, Western Blot, Expressing, Phospho-proteomics