Journal: Frontiers in Pharmacology

Article Title: Retinoic Acid Alleviates Cisplatin-Induced Acute Kidney Injury Through Activation of Autophagy

doi: 10.3389/fphar.2020.00987

Figure Lengend Snippet: RA alleviated CIAKI in Atg5 flox/flox or Cagg-Cre mice. (A) Male and female Atg5 flox/flox , CaggCre, and Atg5 flox/flox :CaggCre mice were divided into four groups randomly (n = 6). Mice were injected with cisplatin with or without RA in different groups as the flowchart showed. (B) Plasma creatinine was measured. (C) Kidney morphology of the cortex and medulla (HE, magnification ×400) showed tubular necrosis (depicted by red arrows) and cast formation (depicted by red asterisks). The RA pretreatment group showed significantly less cast formation than the cisplatin group. Scale bar, 100 μm. (D) Pathological scores of tubular damage in the cortex and medulla of each group. (E) Representative immunofluorescence images of NGAL magnification ×600. NGAL (red), LEL (green), DAPI (blue). Scale bar, 50 μm. (F) Quantification of immunofluorescence images of NGAL. (G) Whole-tissue lysates of kidney were collected for immunoblot analysis of NGAL and β -actin. (H) Densitometry of NGAL signals. Data in B, D, F, and H are expressed as the means ± SDs, *P < 0.05, **P < 0.01, NS, not significant.

Article Snippet: The following primary antibodies were used for immunoblotting or immunofluorescence: rabbit monoclonal anti-Atg5 and mouse anti-p62 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal anti- β -actin (MilliporeSigma, St. Louis, MO, USA), goat monoclonal anti-NGAL (R&D Systems, Minneapolis, MN, USA), rabbit anti-LC3B and anti-Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Injection, Immunofluorescence, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Retinoic Acid Alleviates Cisplatin-Induced Acute Kidney Injury Through Activation of Autophagy

doi: 10.3389/fphar.2020.00987

Figure Lengend Snippet: RA activated autophagy in the kidney in Atg5 flox/flox or Cagg-Cre mice. (A) Whole-tissue lysates of kidney were collected for immunoblot analysis of p62, LC3-II, LC3-I, and β -actin. (B) Quantitative data of the p62 level and LC3-II/LC3-I ratio. Data in B are expressed as the means ± SDs, *P < 0.05, **P < 0.01.

Article Snippet: The following primary antibodies were used for immunoblotting or immunofluorescence: rabbit monoclonal anti-Atg5 and mouse anti-p62 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal anti- β -actin (MilliporeSigma, St. Louis, MO, USA), goat monoclonal anti-NGAL (R&D Systems, Minneapolis, MN, USA), rabbit anti-LC3B and anti-Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Retinoic Acid Alleviates Cisplatin-Induced Acute Kidney Injury Through Activation of Autophagy

doi: 10.3389/fphar.2020.00987

Figure Lengend Snippet: CIAKI was aggravated and the protective effect of RA was attenuated in Atg5 flox/flox :Cagg-Cre mice. (A) Kidney morphology of the cortex and medulla (HE staining, magnification ×400) showed tubular necrosis (depicted by red arrows) and cast formation (depicted by red asterisks). Scale bar, 100 μm. (B) Pathological scores of tubular damage in the cortex and medulla of each group. (C) Blood samples were collected for measurement of plasma creatinine level. Data are expressed as the means ± SDs (n = 6). (D) Whole-tissue lysates of kidney were collected for immunoblot analysis of NGAL and β -actin. (E) Densitometry of NGAL signals. (F) Representative immunofluorescence images of NGAL magnification ×600. NGAL (red), LEL (green), DAPI (blue). Scale bar, 50 μm. (G) Quantification of immunofluorescence images of NGAL of each group. Data in B, C, E, and G are expressed as the means ± SDs, *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant.

Article Snippet: The following primary antibodies were used for immunoblotting or immunofluorescence: rabbit monoclonal anti-Atg5 and mouse anti-p62 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal anti- β -actin (MilliporeSigma, St. Louis, MO, USA), goat monoclonal anti-NGAL (R&D Systems, Minneapolis, MN, USA), rabbit anti-LC3B and anti-Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Western Blot, Immunofluorescence

Journal: Frontiers in Pharmacology

Article Title: Retinoic Acid Alleviates Cisplatin-Induced Acute Kidney Injury Through Activation of Autophagy

doi: 10.3389/fphar.2020.00987

Figure Lengend Snippet: RA inhibited apoptosis after cisplatin-induced RTEC injury in vitro . (A) NRK cells were treated with 0–20 μM cisplatin with or without 1 µM RA for 24 h. Whole-cell lysates of NRK cells were collected for immunoblot analysis of cleaved-caspase-3 and β -actin. (B) Densitometry of cleaved-caspase-3 signals. (C) Cells were fixed with 4% PFA for 10 min and were treated according to the TUNEL kit instructions. Red cells were TUNEL positive cells and the nuclei were stained with DAPI (blue). (D) Quantification of the number of TUNEL-positive cells for every mm 2 under the microscope. (E) Proposed working model of the effect of retinoic acid on autophagy in cisplatin induced AKI. Retinoic acid increases autophagic flux and protects kidney from cisplatin induced injury and exerts antiapoptosis effect. When Atg5 is deficient, the basal autophagy level is downregulated and cisplatin induced AKI is aggravated and the protective effect of retinoic acid is attenuated. Thus retinoic acid protects the kidney against cisplatin-induced injury which is dependent on the regulation of autophagy. Data in B and D are expressed as the means ± SDs, *P < 0.05 **P < 0.01 NS, not significant.

Article Snippet: The following primary antibodies were used for immunoblotting or immunofluorescence: rabbit monoclonal anti-Atg5 and mouse anti-p62 (Novus Biologicals, Littleton, CO, USA), mouse monoclonal anti- β -actin (MilliporeSigma, St. Louis, MO, USA), goat monoclonal anti-NGAL (R&D Systems, Minneapolis, MN, USA), rabbit anti-LC3B and anti-Cleaved Caspase-3 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, Western Blot, TUNEL Assay, Staining, Microscopy

Journal: bioRxiv

Article Title: Activity-dependent post-translational regulation of palmitoylating and de-palmitoylating enzymes in the hippocampus

doi: 10.1101/2022.09.12.507668

Figure Lengend Snippet: (A) Representative images of western blots testing efficacy and specificity of zDHHC and ABHD17 antibodies. Almost all commercially available antibodies for these enzymes were obtained, and the specificity of each antibody was tested. To do this, each zDHHC enzyme tagged with an HA epitope was transfected into HEK 293T cells. The closest phylogenetic or structural zDHHC for each enzyme was also transfected into HEK 293T cells in parallel. The efficacy and specificity of each antibody were then tested using western blotting. We also used Untransfected HEK cells as a negative control. We then tested the antibodies against endogenous proteins in lysates from either rat cultured hippocampal neurons or rat hippocampus Left: HEK cells expressing the indicated tagged zDHHC or ABHD and probed for the tag to demonstrate protein expression. Middle: HEK cells expressing the indicated tagged zDHHC and probed with an antibody from the indicated company. Right: Ability of the antibodies to detect endogenous zDHHCs or ABHDs in rat primary hippocampal cultures or hippocampal lysates. Transfection of zDHHC16, 20, and 23 was unsuccessful. Among all tested antibodies only five (marked with red boxes) were shown to be specific for the target proteins.

Article Snippet: β-actin (1:5000, Sigma A1978), anti ZDHHC1 (1:1000, Abcam ab223042), anti ZDHHC2 (1:1000, Santa Cruz Biotechnology sc-515204), anti ZDHHC2 (1:500, Sigma SAB1101457), anti ZDHHC3 (1:500, Aviva Systems Biology ARP59576), anti ZDHHC3 (1:500, Sigma SAB2107413), anti ZDHHC3 (1:1000, Abcam ab124084),anti ZDHHC3 (1:1000, Abcam ab31837), anti ZDHHC4 (1:500, Aviva Systems Biology ARP78440), anti ZDHHC5 (1:1000 for WB, 5µg for IP, Sigma HPA014670), anti ZDHHC6 (1:600, Abcam ab121423), anti ZDHHC7 (1:500, Aviva Systems Biology OAAB11570), anti ZDHHC7 (1:500, BosterBio A11785), anti ZDHHC8 (1:500, Santa Cruz Biotechnology sc-374191), anti ZDHHC9 (1:1000, Sigma SAB4502104), anti ZDHHC9 (1:1000, Thermo Fisher Scientific PA5-26721), anti ZDHHC11 (1:500, Abcam ab116065), anti ZDHHC12 (1:500, Aviva Systems Biology ARP60674), anti ZDHHC13 (1:500, Aviva Systems Biology ARP44398), anti ZDHHC14 (1:500, Aviva Systems Biology ARP42628), anti ZDHHC15 (1:500, Sigma SAB4500608), anti ZDHHC15 (1:200, Abcam ab121203), anti ZDHHC15 (1:500, Santa Cruz Biotechnology sc-169847), anti ZDHHC15 (1:500, Thermo Fisher Scientific PA5-39327), anti ZDHHC16 (1:500, Aviva Systems Biology ARP50063), anti ZDHHC17 (1:300, Proteintech 15465-1-AP), anti ZDHHC17 (1:500, Sigma AV47141), anti ZDHHC18 (1:1000, Abcam ab154790), anti ZDHHC19 (1:500, Abcam ab179545), anti ZDHHC20 (1:500, Aviva Systems Biology ARP72069), anti ZDHHC21 (1:300, Abcam ab103755), anti ZDHHC22 (1:500, Santa Cruz Biotechnology sc-514005), Phospho-PLK Binding Motif (ST*P) (1:1000, Cell Signaling Technology 5243S), anti-HA (1:1000, Cell Signaling Technology C29F4), anti-myc (1:1000, Cell Signaling Technology 2276), anti-GFP (1:3000, Abcam ab290), anti ABHD17 (1:1000, Origene TA331704), anti ABHD17 (1:1000, Proteintech 15854-1-AP), anti-FLAG (1:1000, Sigma F7425), PSD-95 (1:500, Abcam, ab2723), APT2 (1:500, Abcam, ab151578), TC10 (1:1000, Abcam, ab168645), N-Ras (1:500, Santa Cruz Biotechnology sc-31).

Techniques: Western Blot, Transfection, Negative Control, Cell Culture, Expressing

Journal: bioRxiv

Article Title: Activity-dependent post-translational regulation of palmitoylating and de-palmitoylating enzymes in the hippocampus

doi: 10.1101/2022.09.12.507668

Figure Lengend Snippet: (A) Representative images of fluorescent depalmitoylation probe-5 (DPP-5; 2 μM) 1 hour after cLTP treatment in 14 DIV cultured hippocampal neurons transfected at 11 DIV with mCherry. Left : mCherry cell fill. Middle : Background fluorescence within mCherry mask prior to addition of DPP-5 to the bath. Right : DPP-5 fluorescence within mCherry mask 50 mins post addition of DPP-5 to the bath (1 hr post cLTP). Scale bar = 100 μm. (B) As (A) but 24 hrs post cLTP treatment. (C) Graph of time-course of DPP-5 fluorescence (ΔF) increase following cLTP and subsequent bath addition of DPP-5. No significant difference was observed between mock and cLTP treated neurons at any time-point. (D) Graph of DPP-5 fluorescence (ΔF) 1 hr post cLTP and 50 min post addition of DPP-5 to the bath. No significant difference was observed between mock and cLTP treated neurons at any time-point. (E, F) As (C) and (D) but 24 hrs post cLTP and 50 min post addition of DPP-5 to the bath. ns = not significant (unpaired student’s t -test). Results are mean ± s.e.m. with individual data points shown. N = 14-17 neurons from 2-4 hippocampal cultures. (G) Acyl-Rac assay showing palmitoylated ABHD17 in cultured hippocampal neurons (14 DIV) following cLTP. (H) Phospho-protein purification assay showing phosphorylated ABHD17 following cLTP. (I) Acyl-Rac assay showing palmitoylated APT2 in cultured hippocampal neurons (14 DIV) following cLTP. (J) Phospho-protein purification assay showing phosphorylated APT2 following cLTP. ns = not significant (one-way ANOVA with Tukey’s post hoc test). Results are mean ± s.e.m. with individual data points shown. N = 3 hippocampal cultures per experiment.

Article Snippet: β-actin (1:5000, Sigma A1978), anti ZDHHC1 (1:1000, Abcam ab223042), anti ZDHHC2 (1:1000, Santa Cruz Biotechnology sc-515204), anti ZDHHC2 (1:500, Sigma SAB1101457), anti ZDHHC3 (1:500, Aviva Systems Biology ARP59576), anti ZDHHC3 (1:500, Sigma SAB2107413), anti ZDHHC3 (1:1000, Abcam ab124084),anti ZDHHC3 (1:1000, Abcam ab31837), anti ZDHHC4 (1:500, Aviva Systems Biology ARP78440), anti ZDHHC5 (1:1000 for WB, 5µg for IP, Sigma HPA014670), anti ZDHHC6 (1:600, Abcam ab121423), anti ZDHHC7 (1:500, Aviva Systems Biology OAAB11570), anti ZDHHC7 (1:500, BosterBio A11785), anti ZDHHC8 (1:500, Santa Cruz Biotechnology sc-374191), anti ZDHHC9 (1:1000, Sigma SAB4502104), anti ZDHHC9 (1:1000, Thermo Fisher Scientific PA5-26721), anti ZDHHC11 (1:500, Abcam ab116065), anti ZDHHC12 (1:500, Aviva Systems Biology ARP60674), anti ZDHHC13 (1:500, Aviva Systems Biology ARP44398), anti ZDHHC14 (1:500, Aviva Systems Biology ARP42628), anti ZDHHC15 (1:500, Sigma SAB4500608), anti ZDHHC15 (1:200, Abcam ab121203), anti ZDHHC15 (1:500, Santa Cruz Biotechnology sc-169847), anti ZDHHC15 (1:500, Thermo Fisher Scientific PA5-39327), anti ZDHHC16 (1:500, Aviva Systems Biology ARP50063), anti ZDHHC17 (1:300, Proteintech 15465-1-AP), anti ZDHHC17 (1:500, Sigma AV47141), anti ZDHHC18 (1:1000, Abcam ab154790), anti ZDHHC19 (1:500, Abcam ab179545), anti ZDHHC20 (1:500, Aviva Systems Biology ARP72069), anti ZDHHC21 (1:300, Abcam ab103755), anti ZDHHC22 (1:500, Santa Cruz Biotechnology sc-514005), Phospho-PLK Binding Motif (ST*P) (1:1000, Cell Signaling Technology 5243S), anti-HA (1:1000, Cell Signaling Technology C29F4), anti-myc (1:1000, Cell Signaling Technology 2276), anti-GFP (1:3000, Abcam ab290), anti ABHD17 (1:1000, Origene TA331704), anti ABHD17 (1:1000, Proteintech 15854-1-AP), anti-FLAG (1:1000, Sigma F7425), PSD-95 (1:500, Abcam, ab2723), APT2 (1:500, Abcam, ab151578), TC10 (1:1000, Abcam, ab168645), N-Ras (1:500, Santa Cruz Biotechnology sc-31).

Techniques: Cell Culture, Transfection, Fluorescence, Protein Purification