Journal: The Journal of Biological Chemistry

Article Title: UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase *

doi: 10.1074/jbc.M116.766824

Figure Lengend Snippet: Alignment of UBE3B with select IQ motif proteins and HECT E3 ubiquitin ligases. A, schematic of UBE3B showing the IQ domain (amino acids 29–58) and the HECT domain (amino acids 757–1068). The proposed 3D structures of the IQ and HECT domains using Phyre2 are shown above the schematic. The N terminus of HECT domains are known to bind to substrate. The HECT domain is composed of two lobes as follows: the N-lobe binds the E2(s), and the C-lobe contains the catalytic cysteine that binds ubiquitin. B, alignment of UBE3B with calmodulin binding domains as predicted by Phyre2 and using ClustalW2. C, alignment of UBE3B with HECT E3 ligase domains as predicted by Phyre2 and using ClustalW2. The conserved catalytic cysteine is highlighted in red. * denotes a single fully conserved residue; : denotes conservation between groups of strongly similar properties, . denotes conservation between groups of weakly similar properties.

Article Snippet: UBE3B antibody (catalog no. SAB4503523) was from Sigma; ubiquitin (Ub) antibody (catalog no. AUB01) was from Cytoskeleton; α-tubulin antibody (catalog no. CP06) was from EMD Millipore; proliferating cell nuclear antigen antibody (catalog no. sc-56) was from Santa Cruz Biotechnology; cytochrome c oxidase IV (COX IV) antibody (catalog no. A21348) was from Life Technologies, Inc.; Turbo-green fluorescent protein (TurboGFP) antibody (catalog no. AB513) was from Evrogen; adenosine triphosphate synthase β (ATP synthase β) antibody (catalog no. MA1-930) was from Thermo Fisher Scientific; calmodulin antibody (catalog no. ab45689) was from Abcam; PDI antibody was from Life Technologies, Inc. (catalog no. RL77); and cleaved caspase 3 antibody was from Cell Signaling (catalog no. 9661s).

Techniques: Ubiquitin Proteomics, Binding Assay, Residue

Journal: The Journal of Biological Chemistry

Article Title: UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase *

doi: 10.1074/jbc.M116.766824

Figure Lengend Snippet: UBE3B exhibits ubiquitin ligase activity. Cell lysates from LN428 cells that stably express UBE3A, UBE3B, UBE3BΔHECT, or UBE3B(C1036A), all with N terminus HA tags, were immunoprecipitated using anti-HA affinity matrix and subjected to ubiquitylation activity assays. The ubiquitylation assays were carried out in 30-μl reactions with 0.1 μm E1, 0.25 μm of an E2 mixture, 1 μm ubiquitin aldehyde, 0.75 μg/μl His-ubiquitin, and 1× magnesium/ATP mixture, unless otherwise noted. A, ubiquitylation activity assay was performed in vitro using His-tagged wild type ubiquitin (His-Ub-WT). The expected size of HA-UBE3B is 125 kDa. Note the time-dependent increase in the high molecular mass signal. B, polyubiquitin chain formation was lost when ubiquitin or E1, E2s, or ATP were removed from the reaction mixture, or if 20 μg of the catalytic core of the deubiquitinating enzyme USP2 was added to a completed reaction for 30 min at 37 °C (63). C, polyubiquitin chains were lacking in reactions containing the catalytically inactive (HA-UBE3B(C1036A)) or HECT deleted (HA-UBE3BΔHECT) mutants of UBE3B. Completed reactions were applied to immunoblot for analysis of ubiquitylation with anti-hemagglutinin (HA) or ubiquitin (Ub) antibodies. Bottom panel shows comparable loading of the immunoprecipitated proteins. Representative immunoblots from three independent experiments are shown. Antibodies used for each of the immunoblots are listed on the side of the panels.

Article Snippet: UBE3B antibody (catalog no. SAB4503523) was from Sigma; ubiquitin (Ub) antibody (catalog no. AUB01) was from Cytoskeleton; α-tubulin antibody (catalog no. CP06) was from EMD Millipore; proliferating cell nuclear antigen antibody (catalog no. sc-56) was from Santa Cruz Biotechnology; cytochrome c oxidase IV (COX IV) antibody (catalog no. A21348) was from Life Technologies, Inc.; Turbo-green fluorescent protein (TurboGFP) antibody (catalog no. AB513) was from Evrogen; adenosine triphosphate synthase β (ATP synthase β) antibody (catalog no. MA1-930) was from Thermo Fisher Scientific; calmodulin antibody (catalog no. ab45689) was from Abcam; PDI antibody was from Life Technologies, Inc. (catalog no. RL77); and cleaved caspase 3 antibody was from Cell Signaling (catalog no. 9661s).

Techniques: Ubiquitin Proteomics, Activity Assay, Stable Transfection, Immunoprecipitation, In Vitro, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase *

doi: 10.1074/jbc.M116.766824

Figure Lengend Snippet: UBE3B associates with calmodulin through the IQ motif in a calcium-dependent manner. A, quantification results of HA-Ab affinity purification and high resolution LC-MS analysis for two proteotypic calmodulin peptides, VFDKDGNGYISAAELR and DTDSEEEIREAFR. Bar graphs represent the standardized mean values ± S.E. following affinity purification and differential mass spectrometry analysis of LN428 cells containing an empty vector control or expressing wild type HA-tagged UBE3B (HA-UBE3B) or the catalytic inactive UBE3B with an HA tag (HA-UBE3B(C1036A)). B, cells, indicated in the figure, were lysed, and the HA-tagged proteins were immunoprecipitated, as described under “Experimental Procedures,” to identify the proteins that interact with UBE3B, UBE3B(C1036A), or UBE3BΔHECT. Shown is an immunoblot to confirm the interaction of calmodulin with UBE3B as seen by LC-MS using anti-UBE3B and calmodulin antibodies. C, BioID analysis was performed using BirA-UBE3B and UBE3B-BirA with streptavidin immunoprecipitated to confirm that HA-UBE3B and calmodulin interact. Cells were treated with biotin (5 μm) for 24 h before harvest, as indicated. Streptavidin-purified proteins were then probed by immunoblot using streptavidin-HRP. Bottom right panel indicates the identification of calmodulin after streptavidin capture. D, 293FT cells were transiently transfected (48 h) with a plasmid expressing WT UBE3B-HA or UBE3BΔIQ-HA followed by immunoprecipitation with HA-affinity matrix beads. Deletion of the IQ motif demolished the interaction between UBE3B and calmodulin. E, 239FT cells were transiently transfected (48 h) with a plasmid expressing WT UBE3B-HA or UBE3BΔIQ-HA followed by immunoprecipitation with HA-affinity matrix beads. A ubiquitylation assay was then performed as described under “Experimental Procedures.” The results were analyzed by immunoblot using anti-ubiquitin, anti-His, and anti-HA antibodies to detect UBE3B and anti-CaM antibody to detect calmodulin. Ubiquitylation is increased in UBE3BΔIQ due to the loss of loss of the interaction with calmodulin. F, 239FT cells were transiently transfected (48 h) with a plasmid expressing WT UBE3B-HA or UBE3BΔIQ-HA. The expression of UBE3BΔIQ-HA triggered cell apoptosis as indicated by an elevation of the cleaved form of caspase3. G, calmodulin interaction with UBE3B is lost when immunoprecipitation beads are washed with CaCl2, and ubiquitylation is increased when calmodulin is not bound to UBE3B. Immunoprecipitations and the bead-based in vitro assay were performed as described above.

Article Snippet: UBE3B antibody (catalog no. SAB4503523) was from Sigma; ubiquitin (Ub) antibody (catalog no. AUB01) was from Cytoskeleton; α-tubulin antibody (catalog no. CP06) was from EMD Millipore; proliferating cell nuclear antigen antibody (catalog no. sc-56) was from Santa Cruz Biotechnology; cytochrome c oxidase IV (COX IV) antibody (catalog no. A21348) was from Life Technologies, Inc.; Turbo-green fluorescent protein (TurboGFP) antibody (catalog no. AB513) was from Evrogen; adenosine triphosphate synthase β (ATP synthase β) antibody (catalog no. MA1-930) was from Thermo Fisher Scientific; calmodulin antibody (catalog no. ab45689) was from Abcam; PDI antibody was from Life Technologies, Inc. (catalog no. RL77); and cleaved caspase 3 antibody was from Cell Signaling (catalog no. 9661s).

Techniques: Affinity Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Plasmid Preparation, Control, Expressing, Immunoprecipitation, Western Blot, Purification, Transfection, Ubiquitin Assay, Ubiquitin Proteomics, In Vitro

Journal: The Journal of Biological Chemistry

Article Title: UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase *

doi: 10.1074/jbc.M116.766824

Figure Lengend Snippet: Oligodeoxynucleotides used in this study F is forward; R is reverse.

Article Snippet: UBE3B antibody (catalog no. SAB4503523) was from Sigma; ubiquitin (Ub) antibody (catalog no. AUB01) was from Cytoskeleton; α-tubulin antibody (catalog no. CP06) was from EMD Millipore; proliferating cell nuclear antigen antibody (catalog no. sc-56) was from Santa Cruz Biotechnology; cytochrome c oxidase IV (COX IV) antibody (catalog no. A21348) was from Life Technologies, Inc.; Turbo-green fluorescent protein (TurboGFP) antibody (catalog no. AB513) was from Evrogen; adenosine triphosphate synthase β (ATP synthase β) antibody (catalog no. MA1-930) was from Thermo Fisher Scientific; calmodulin antibody (catalog no. ab45689) was from Abcam; PDI antibody was from Life Technologies, Inc. (catalog no. RL77); and cleaved caspase 3 antibody was from Cell Signaling (catalog no. 9661s).

Techniques: Sequencing, Ubiquitin Proteomics

Journal: Frontiers in Cellular Neuroscience

Article Title: Adipose-derived stem cell-conditioned medium mitigates ischemia-induced neuronal injury via the JAK1/STAT3 signaling pathway

doi: 10.3389/fncel.2026.1744887

Figure Lengend Snippet: ADSC-CM promotes neurite outgrowth in OGD-injured neurons through the JAK1-STAT3 signaling pathway. (A) On day 5 of cultivation, neuronal neurites extended and formed an extensive neural network. Immunofluorescence staining showed that the neurons were positive for the neuronal-specific marker Tuj1. After OGD injury, neuronal neurites were damaged, partially interrupted, and disappeared. (B) Western blot bands of JAK1, pJAK1, STAT3, pSTAT3, and GAPDH for each group. (C) Comparison of the relative ratio of pJAK1 to JAK1 across experimental groups ( n = 4). (D) Comparison of the relative ratio of pSTAT3 to STAT3 across experimental groups ( n = 4). (E) Immunofluorescence images of neurons from each group, showing the effects of ADSC-CM and GLPG0634 on neurite outgrowth. Neurons were stained with the Tuj1 antibody (red) to label the neuronal cytoskeleton, and nuclei were counterstained with DAPI (blue). (F) Diagram illustrating how to measure the length of the longest neurite and the number of primary neurites in neurons. The green line shows the trajectory of the longest neurite, and white arrows point to primary neurites. (G,H) Comparison of the length of the longest neurite and the number of primary neurites in neurons across experimental groups ( n = 4). Data are expressed as means ± SEM. The difference between the groups was assessed using a one-way ANOVA followed by Bonferroni post hoc tests. * p < 0.05, ** p < 0.01, *** p < 0.001. Scale bars: 100 μm (A) , 20 μm (E,F) .

Article Snippet: After overnight incubation with rabbit anti-Tuj1 antibody (1:200, BM3881, Boster, Wuhan, China) at 4 °C, cells were washed and then incubated with Cy3-conjugated goat anti-rabbit IgG (1:400, AS007, ABclonal) for 1 h at room temperature in the dark.

Techniques: Immunofluorescence, Staining, Marker, Western Blot, Comparison

Journal: Scientific Reports

Article Title: Blocking protein phosphatase 2A signaling prevents endothelial-to-mesenchymal transition and renal fibrosis: a peptide-based drug therapy

doi: 10.1038/srep19821

Figure Lengend Snippet: ( a ) Representative immunohistochemical detection of PP2Ac in the kidney tissue of sham-operated and UUO mice (left). Co-localization of PP2Ac (green) with CD31(red) was visualized as yellow in the merged images from sham-operated and UUO-14d mice (n = 8). Scale bar: 20 μm. ( b,c ) Western blot analysis of PP2Ac expression in sham-operated and UUO-14d mice (n = 8) and in TGF-β1-treated HUVECs for the indicated periods with densitometry analysis (n ≥ 3). ( d ) HUVECs were incubated with or without TGF-β1 for 72 h and were harvested for immunoprecipitation analysis. Cell lysates were subjected to immunoprecipitation (IP) with an anti-PP2Ac anti-body, followed by immunoblotting (IB) with anti-3-nitrotyrosine (3-NT), anti-acetylation (acetyl), anti-phosphotyrosine (p-Tyr), and anti-methylation (unmethylated) antibodies, respectively. Densitometry analysis of 3-NT, acetyl, p-Tyr and methyl of PP2Ac in d. ( e ) Analysis of PP2A activity in immunoprecipitation of 3-NT-IP, Acetyl-IP, p-Tyr-IP and Unmethylated-IP, expressed as percentage of the value in control group, respectively (n ≥ 3). *P < 0.05 versus the basal condition. NS: no significance. Bars represent means ± sd.

Article Snippet: After transfer, the membranes were blocked and blotted routinely with antibodies against VE-cadherin (BD Biosciences), α-SMA (Abcam), collagen1 (Abcam), PP2Ac (Cell Signaling Technology), occludin (Invitrogen), P-threonine/P-serine (Santa Cruz), nitrotyrosine (Cayman), acetylated-lysine (Cell Signaling Technology), phosphotyrosine (PY-20,Biolgend), PP2A,C subunit, demethylated (Millipore), vimentin (Boster) and CD31 (Proteintech).

Techniques: Immunohistochemical staining, Western Blot, Expressing, Incubation, Immunoprecipitation, Methylation, Activity Assay, Control

Journal: Genetics

Article Title: DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae

doi: 10.1534/genetics.116.196568

Figure Lengend Snippet: Srs2 is a target of Uls1 activity. (A) Uls1 regulates the protein level of Srs2 helicase. Wild type (WT) (W303-1A), uls1Δ (MC001), and ADH1-HA-ULS1 (MC037) strains were transformed with CUP1-SRS2 (pWJ1509) or CUP1-srs2-R1 (pAM19) plasmids and grown to logarithmic phase in selective medium followed by 4-hr incubation in the presence of 200 μM CuSO4 to induce overexpression of SRS2 alleles. Then cells were collected for protein extraction and Western blot analysis using anti-Srs2 antibody. The srs2Δ mutant (MC010) was included in the analysis as a control of antibody specificity. The signal detected for Srs2 vs. total protein as determined by Ponceau-S staining was quantified for three experiments. (B) SUMOylation status of Srs2 is affected by activity of Uls1. Aliquots of protein extracts obtained in (A) were used for IP reaction with anti-Srs2 antibody. Immunoprecipitates were analyzed by immunoblotting with anti-Srs2 and anti-SUMO antibodies. Srs2-SUMO levels vs. total immunoprecipitated Srs2 from two experiments were quantified. SDs are shown and Student’s t-test was used to calculate the P-value (* 0.01 < P-value ≤ 0.05, ** 0.001 < P-value ≤ 0.01).

Article Snippet: After visualizing Srs2 protein, the membrane was stripped with 0.4 M NaOH and probed with anti-SUMO antibody (200-401-428; Rockland) to detect the SUMOylated forms of Srs2 protein.

Techniques: Activity Assay, Transformation Assay, Incubation, Over Expression, Protein Extraction, Western Blot, Mutagenesis, Control, Staining, Immunoprecipitation

Journal: Genetics

Article Title: DNA Damage Tolerance Pathway Choice Through Uls1 Modulation of Srs2 SUMOylation in Saccharomyces cerevisiae

doi: 10.1534/genetics.116.196568

Figure Lengend Snippet: Comparison of the effect of SLX5 and ULS1 deletion on the accumulation of SUMOylated Srs2. (A) Wild type (WT) (W303-1A), uls1Δ (MC001), and slx5Δ (KK013) strains were transformed with CUP1-SRS2 (pWJ1509) and after overexpression of SRS2 (as in Figure 2), protein extracts were isolated and Western blot analysis with anti-Srs2 antibody was performed. The signal detected for Srs2 vs. total protein as determined by Ponceau-S staining was quantified for two experiments. (B) IP of Srs2 from extracts obtained in (A) and visualization of SUMOylated Srs2 using anti-Srs2 and anti-SUMO antibody. Srs2-SUMO levels vs. total immunoprecipitated Srs2 from two experiments were quantified. (C) Uls1 and Slx5 mediate turnover of the Srs2 protein. Endogenous Srs2 level was analyzed by CHX chase. Wild type (W303-1A), uls1Δ (MC001), and slx5Δ (KK013) were synchronized in G1 and released into YPD medium containing 0.03% MMS and 50 μg/ml CHX. At the indicated time points, cells were collected for protein extraction followed by Western blotting with the anti-Srs2 antibody. Level of G-6-PDH was used as a loading control. The srs2Δ mutant (MC010) was included in the analysis as a control of antibody specificity. The percentage values of relative Srs2 level vs. G-6-PDH, acquired for two experiments, are plotted. SDs are shown and Student’s t-test was used to calculate the P-value (* 0.01 < P-value ≤ 0.05).

Article Snippet: After visualizing Srs2 protein, the membrane was stripped with 0.4 M NaOH and probed with anti-SUMO antibody (200-401-428; Rockland) to detect the SUMOylated forms of Srs2 protein.

Techniques: Comparison, Transformation Assay, Over Expression, Isolation, Western Blot, Staining, Immunoprecipitation, Protein Extraction, Control, Mutagenesis

Journal: Journal of Thoracic Disease

Article Title: Isoforskolin inhibits LUBAC/GSDMD/IL-1β cascades in pulmonary fibrosis

doi: 10.21037/jtd-2025-1950

Figure Lengend Snippet: ISOF inhibited the expression of LUBAC in MLGs. (A) The protein levels of SHARPIN in the MLGs stimulated with TGF-β1 was analyzed by western blot. The western blot representative strip was listed on the right of figure. (B) The protein levels of HOIL-1L in the MLGs stimulated with TGF-β1 was analyzed by western blot. The western blot representative strip was listed on the right of figure. (C) The protein levels of HOIP in the MLGs stimulated with TGF-β1 was analyzed by western blot. The western blot representative strip was listed on the right of figure. ANOVA was used for western blotting statistics (SHARPIN, n=8; HOIL-1L, n=5; HOIP, n=10). ANOVA, analysis of variance; DXM, dexamethasone; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HOIL-1L, heme oxidation IRP2 ubiquitin ligase 1L; HOIP, HOIL-1L interacting protein; HOIPIN-8, HOIP inhibitor-8; ISOF, isoforskolin; LUBAC, linear ubiquitin chain assembly complex; MLGs, mice lung fibroblasts; PFD, pirfenidone; SHARPIN, shank-associated RH domain interaction protein; TGF-β1, transforming growth factor-β1.

Article Snippet: Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11, XP ® Rabbit mAb#5174), IL-1β (D6D6T, Rabbit mAb#31202), GSDMD (E9S1X, Rabbit mAb#39754), beta-Actin (13E5, Rabbit mAb#4970) antibodies were derived from Cell Signaling Technology (Danvers, MA, USA), antibodies against HOIP (#A04457-3), SHARPIN (#A06687-1), and beta-Tubulin (#A05397-1) were purchased from Boster Biological (Wuhan, China).

Techniques: Expressing, Western Blot, Stripping Membranes, Ubiquitin Proteomics

Journal: Journal of Thoracic Disease

Article Title: Isoforskolin inhibits LUBAC/GSDMD/IL-1β cascades in pulmonary fibrosis

doi: 10.21037/jtd-2025-1950

Figure Lengend Snippet: ISOF reduced the expression level of LUBAC in IPF model mice. (A) The protein levels of SHARPIN in the lung tissues of each group was assessed by western blot. (B) The protein levels of HOIL-1L in the lung tissues of each group was assessed by western blot. (C) The protein levels of HOIP in the lung tissues of each group was assessed by western blot. The relative density of LUBAC subunits to GAPDH was calculated in each group. Data were expressed as means ± SEM (SHARPIN, n=7; HOIL-1L, n=8; HOIP, n=7). BLM, bleomycin; DXM, dexamethasone; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HOIL-1L, heme oxidation IRP2 ubiquitin ligase 1L; HOIP, HOIL-1L interacting protein; IPF, idiopathic pulmonary fibrosis; ISOF, isoforskolin; LUBAC, linear ubiquitin chain assembly complex; PFD, pirfenidone; SEM, standard error of measurement; SHARPIN, shank-associated RH domain interaction protein.

Article Snippet: Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (D16H11, XP ® Rabbit mAb#5174), IL-1β (D6D6T, Rabbit mAb#31202), GSDMD (E9S1X, Rabbit mAb#39754), beta-Actin (13E5, Rabbit mAb#4970) antibodies were derived from Cell Signaling Technology (Danvers, MA, USA), antibodies against HOIP (#A04457-3), SHARPIN (#A06687-1), and beta-Tubulin (#A05397-1) were purchased from Boster Biological (Wuhan, China).

Techniques: Expressing, Western Blot, Ubiquitin Proteomics

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Sequences of primers.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Sequencing, Amplification

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA and FGF2 are upregulated in pancreatic cancer tissues and cell lines. (A) Comparison of HPA and FGF2 expression in pancreatic cancer tissues and adjacent normal pancreatic tissues by immunohistochemical stainin. (B) Analysis of relative mRNA expression levels of HPA and FGF2 in pancreatic cancer and normal pancreatic cell lines by reverse transcription-quantitative PCR. *P<0.05, **P<0.01 vs. HPDE6c7. HPA, heparanase; FGF2, fibroblast growth factor 2; NC, negative control.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Comparison, Expressing, Immunohistochemical staining, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Expression of heparanase and fibroblast growth factor 2 in pancreatic cancer tissues and adjacent normal tissues.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: Association between heparanase and fibroblast growth factor 2 expression, and clinicopathological characteristics of patients with pancreatic cancer.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA upregulates the expression of FGF2 in pancreatic cancer cell lines. (A) Analysis of HPA silencing and overexpression efficiency by RT-qPCR. (B) Analysis of HPA silencing and overexpression efficiency by western blot assay. (C) Analysis of FGF2 expression after silencing or overexpressing HPA by RT-qPCR. (D) Analysis of FGF2 expression after silencing or overexpressing HPA by western blotting. β-actin was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. RT-qPCR, reverse transcription-quantitative PCR; HPA, heparanase; FGF2, fibroblast growth factor 2; sh, short hairpin (RNA; specific for HPA); Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Transfection, Sequencing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA regulates FGF2 expression via the HPA/SDC1 axis in pancreatic cancer cell lines. (A) RT-qPCR comparing the relative expression levels of SDC1 mRNA in pancreatic cancer lines and the normal pancreatic cell line HPDE6c7. *P<0.05, **P<0.01 vs. HPDE6c7. (B) Detection of the effect of HPA on SDC1 mRNA expression by RT-qPCR. (C) Expression of FGF2 was determined by RT-qPCR analysis after adding the SDC1 inhibitor synstatin to the HPA overexpression group. **P<0.01. RT-qPCR, reverse transcription-quantitative PCR; HPA, heparanase; FGF2, fibroblast growth factor 2; sh, short hairpin (RNA); SDC1, syndecan-1; Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, shRNA, Plasmid Preparation, Transfection, Sequencing

Journal: Oncology Letters

Article Title: The HPA/SDC1 axis promotes invasion and metastasis of pancreatic cancer cells by activating EMT via FGF2 upregulation

doi: 10.3892/ol.2019.11121

Figure Lengend Snippet: HPA promotes cell migration and invasion by activating the PI3K/Akt signaling pathway and EMT processes via FGF2 upregulation. (A) Expression of Palladin protein in various pancreatic cancer cell lines was analyzed by western blot assay. β-actin was used as an internal control. (B) Analysis of the relative expression of Palladin mRNA in pancreatic cancer cell lines by reverse transcription-quantitative PCR. (C) Western blot analysis of the effect of FGF2 on Palladin and Akt. (D) Protein expression of Palladin and Akt. β-actin was used as an internal control. (E) Effect of FGF2, AZD4547 and LY294002 on the migratory ability of PANC-1 cells assessed by wound healing assay. Magnification, ×200. (F) Transwell assays were performed to determine the invasive ability of PANC-1 cells treated with FGF2, AZD4547 and LY294002. (G) Number of invaded PANC-1 cells treated with FGF2, AZD4547 and LY294002. CON, PANC-1 cell line transfected with empty plasmid. (H) Western blot analysis of the effect of HPA and FGF2 on EMT. Vector, PANC-1 cell line transfected with empty plasmid. β-actin was used as an internal control. *P<0.05, **P<0.01, ***P<0.001. HPA, heparanase; FGF2, fibroblast growth factor 2; EMT, epithelial-mesenchymal transition; CON, PANC-1 cell line transfected with empty plasmid; Vector, PANC-1 cell line transfected with empty plasmid containing the GFP sequence.

Article Snippet: After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated with the following primary antibodies: Rabbit anti-human HPA (1:1,000; cat. no. PB0405), rabbit anti-human FGF2 (1:1,000; cat. no. PB0916), rabbit anti-human AKT (1:5,000; cat. no. A00024-1), rabbit anti-human E-cadherin (1:100; cat. no. BA0475), rabbit anti-human N-cadherin (1:100; cat. no. BM3921), rabbit anti-human vimentin 1:3,000 (cat. no. PB0378; all Boster Biological Technology) and rabbit anti-human palladin (1:100; cat. no. PA5-65160; Thermo Fisher Scientific Inc.) were added according to the instructions of the manufacturer and incubated at 4°C overnight. β-actin (1:500; cat. no. BA2305; Boster Biological Technology) was used as an internal loading control.

Techniques: Migration, Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Wound Healing Assay, Transfection, Plasmid Preparation, Sequencing

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Cosmo Bio anti-LC3B specifically recognizes LC3B in EGFP-LC3B transfected cells. (A) Schematic overview of endo-lysosomal and autophagic compartments, outlining their characteristic morphological features and presence of LC3. (B-C) EGFP-LC3B transfected cells were fixed with 2% PFA+0.2% GA for 3 h and double labeled for LC3B and GFP and protein A gold (PAG). (B) The labeling intensities of anti-GFP (1:400; PAG15) and Cosmo Bio anti-LC3B (1:10; PAG10) correlate in cells with high and low EGFP-LC3B overexpression. (C) Example of double labeling with anti-GFP (1:400; PAG15) and another LC3 antibody (CST, 4108, 1:15; PAG10) showing only PAG15 on EGFP-LC3B overexpressing cells. AL, autolysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 300 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Transfection, Labeling, Over Expression, Clinical Proteomics, Membrane

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Performance of commercial LC3 antibodies in IF and immuno-EM.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques:

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Imaging autophagy by conventional EM and immunofluorescence. (A) Conventional EM of control U2OS cells. Endo-lysosomal compartments are identified by morphology. (B) Conventional EM of U2OS cells starved for 2.5 h in the presence of BafA1 showing an accumulation of different types of autophagic compartments. Autophagosomes (AP) are recognized by their double-membrane that encapsulates part of the cytoplasm with the same density as the surrounding cytoplasm and containing multiple membrane structures. Autolysosomes (AL) are vacuoles lined by a single membrane. Their lumen is heterogenous in content (membranes, vesicles, amorphous material) and electron density (from light to dense). Both AP and AL contain recognizable endoplasmic reticulum (ER) cisternae (arrows). Asterisks indicate autophagic content. (C-E) IF of semi-thin cryosections of control and starved, BafA1-treated U2OS cells, fixed either with 4% PFA ON (PFA) or 2% PFA+0.2% GA for 2 h (PFA+GA) and labeled with Cosmo Bio anti-LC3B (1:10) and Alexa Fluor 488-conjugated goat anti-mouse IgG (1:300). Images are recorded with identical settings and represented with equal intensity ranges. PFA and PFA+GA fixed cells contain comparably intense LC3B puncta. AL, autolysosome; AP, autophagosome; E, endosome; Ly, lysosome; M, mitochondrion; PM, plasma membrane. Scale bars: 500 nm (A-B); 20 µm (C-E).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Imaging, Immunofluorescence, Control, Membrane, Labeling, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Correlative light electron microscopy (CLEM) of endogenous LC3B on ultrathin cryosections. (A) Ultrathin (60–70 nm) cryosection of starved, BafA1-treated U2OS cells (fixation 4% PFA ON) labeled for LC3B (1:10), rabbit anti-mouse IgG, Alexa Fluor 488-conjugated donkey anti-rabbit IgG and PAG10, showing IF puncta (green) and nuclei (Hoechst, blue). (B) Low magnification EM picture of same section as in (A). Arrows point to LC3B-immunogold labeled compartments (gold not visible at this magnification). (C) Overlay of the fluorescent and EM images in (A) and (B). Higher magnifications of the boxed areas in (C) are shown in (D-F). (D) A phagophore (PG) is visible as a ring of LC3B-positive vesicles. Arrows indicate visible membrane contours. (E, F) Two examples of autolysosomes (AL). LC3B (PAG10) is predominantly associated with autophagic content located in the AL lumen (indicated by asterisks). Note that the higher magnifications in D-F are rotated relative to B. N, nucleus. Scale bars: 2 µm (A-C), 200 nm (D-F).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Electron Microscopy, Labeling, Membrane

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Effect of different fixation and labeling regimes on LC3 immuno-EM labeling efficiency. (A-D) Electron micrographs of U2OS cells starved in the presence of BafA1 for 2.5 h. Cells were fixed and immunolabeled for LC3B (1:10; PAG10) according to the standard or fast labeling protocol as indicated. (A, B) Cells fixed ON with 4% PFA labeled by the standard (A) or fast (B) protocol. Both conditions show abundant LC3B label on autophagic content (asterisks) present in autolysosomes (AL). (C) Cells fixed for 15 min with 4% PFA, followed by 6 days of 0.6% PFA and labeled by the fast protocol. (D) Cells fixed for 3 h with 2% PFA+0.2% GA labeled by the standard protocol. Note a reduction in LC3B label compared to A-C. (E) Quantification of the number of LC3B-representing PAG10 particles per organelle for the conditions shown in A-D. Cells were randomly screened for LC3B-positive organelles. The number of organelles (N) screened was 80, 71, 74 and 25 for conditions indicated in A, B, C and D, respectively. Only the PFA+GA condition significantly differs from the others at p ≤ 0.0001 using Student’s t -test assuming unequal variance (***). (F) Starved, BafA1-treated U2OS cells fixed with 4% PFA for 15 min followed by 6 days with 0.6% PFA. Representative image of an autophagosome (AP) labeled for LC3B (1:6) using a gold enhancement step after PAG5 labeling. AP, autophagosome; ER, endoplasmic reticulum; M, mitochondrion. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Immunolabeling

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Effect of different fixation and labeling regimes on morphology. U2OS cells starved for 2.5 h in the presence of BafA1, fixed and labeled with Cosmo Bio anti-LC3B and PAG10 according to the indicated fixation and labeling protocols. ( A, E, G ) Typical examples of autophagosomes (AP) using different fixation and labeling regimes. The double membrane is partially extracted resulting in a halo with remnants of inner (open arrowheads) and outer (black arrowheads) membrane. Arrows indicate LC3 PAG10 associated with the outer membrane. (C) Phagophore (PG), recognizable by the edge (shafted arrowhead) of the cup-shaped double membrane. ( B, D, F, H ) Examples of autolysosomes (AL) using the indicated fixation and labeling regimes. Autolysosomes typically contain autophagic content (asterisks) positive for LC3B, are sometimes filled with internal vesicles and display an overall heterogeneous content. The different PFA fixations yield comparable morphologies. The PFA+GA fixation yields an overall better ultrastructure, yet still results in the halo around phagophores and autophagosomes. Dilutions Cosmo Bio LC3B antibody: 1:4 (A, C, D), 1:10 (B, E-H). Asterisk, autophagic content. ER, endoplasmic reticulum; G, Golgi; M, mitochondrion; N, nucleus; PM, plasma membrane. Scale bars: 100 nm (A, C, E, G, H), 200 nm (B, D, F).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Membrane, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: Immuno-EM of endogenous LC3B in primary cells and tissues. (A-B) Primary mouse macrophages were starved for 30 min without addition of BafA1. LC3B labeling (1:6; PAG10, arrows) is detected on autophagosomes (AP) and autolysosomes (AL). Gold particles are associated with the inner (open arrowheads) and outer (black arrowheads) autophagosome membrane. (A) Cells fixed ON with 4% FPA. (B) Cells fixed with 2% PFA+0.2% GA for 2 h. (C-H) In rat pancreas and liver (1:10; PAG10), in the absence of lysosomal inhibitors, LC3B labeling is detected mainly on autophagosomes (AP) and occasionally on an autolysosome (AL). Gold particles are associated with the inner and outer (arrows) autophagosome membrane. (C-F) Rat exocrine pancreas perfusion fixed with 2% PFA+0.2% GA. (C) Overview. (D, E) Examples of autophagosomes (AP) encapsulating mainly ER membranes. (F) Group of 2 (not-labeled) autolysosomes (AL) and an autophagosome (AP) near the Golgi (G), enlarged from the box in (C). LC3 immunogold label is present on inner and outer (arrows) AP membrane. (G, H) Rat liver perfusion fixed with 4% PFA. (H) LC3 immunogold label on a group of autophagosomes (AP) and an autolysosome (AL) in a hepatocyte, enlarged from the box in (G). Arrows indicate LC3 gold on the outer AP membrane. BC, bile canaliculus; EE, early endosome; ER, endoplasmic reticulum; M, mitochondrion; NE, nuclear envelope; PM, plasma membrane; SG, secretory granule. Scale bars: 100 nm (D), 200 nm (A, B, E, F, H), 1 μm (C), 500 nm (G).

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Labeling, Membrane, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: LC3B colocalizes with endocytosed BSA 5 , LAMP1 and SQSTM1/p62 in autolysosomes of starved, BafA1-treated U2OS cells. (A) Timeline of experimental setup. (B) Epon section showing BSA 5 -containing autolysosome (AL) with similar morphological features as the AL in (C). (C) LC3B (1:4; PAG10) is present in an autolysosome (AL) also containing BSA 5 (black arrowheads). White arrowheads mark the limiting membrane of the AL, showing that LC3B and BSA 5 colocalize in the AL lumen. (D) Double labeling of LAMP1 (1:60; PAG10) and LC3B (1:10; PAG15). Accumulation of LC3B inside a LAMP1-positive autolysosome (AL) also positive for endocytosed BSA 5 (black arrowheads). (E) Colocalization of LC3B (1:6; PAG15) and SQSTM1/p62 (1:100; PAG10) in a typical autolysosome (AL). SQSTM1/p62 label (arrows) marks only a subset of the LC3B-positive material. Fixation for immuno-EM: (C) and (E) 4% PFA ON; (D) 2% PFA, 0.2% GA, 3 h. Asterisks, autophagic content. PM, plasma membrane. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Membrane, Labeling, Clinical Proteomics

Journal: Autophagy

Article Title: An optimized protocol for immuno-electron microscopy of endogenous LC3

doi: 10.1080/15548627.2022.2056864

Figure Lengend Snippet: LC3B is incorporated in autolysosomes after extended incubation with BafA1. U2OS cells were treated with BafA1 for the indicated durations and starved in presence of BafA1 using EBSS for 2.5 h before fixation with 4% PFA. Double labeling of LC3B (1:10; PAG15) and LAMP1 (1:100; PAG10) reveals that in all conditions the majority of LC3B label is found in LAMP1-positive autolysosomes (AL). (A) 2.5 h; (B) 5 h; (C) 10 h; (D) 24 h of BafA1 incubation. (E) Quantification of >50 LC3B-positive organelles scored for presence of LAMP1 label per condition. These results indicate that fusion of LC3B-positive autophagosomes with lysosomes proceeds after BafA1 treatment. For more examples of LC3B labeling after BafA1 treatment and the effect on autolysosome morphology, see Fig. S3. Asterisks, autophagic content. M, mitochondrion; N, nucleus. Scale bars: 200 nm.

Article Snippet: For the LC3-GFP double labeling, LC3 was first labeled with LC3 antibody, secondary rabbit anti-mouse IgG (in case of a mouse anti-LC3 antibody) and PAG10, followed by GFP labeling either with biotinylated anti-GFP antibody (Rockland), rabbit anti-biotin and PAG15, or with rabbit anti-GFP (Thermo Fisher Scientific) and PAG15.

Techniques: Incubation, Labeling