Journal: FEBS Open Bio

Article Title: Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing

doi: 10.1002/2211-5463.13380

Figure Lengend Snippet: RNA interference and ROS detection. (A) The experimental formulas of achieved 70% transfection efficiency of shRNA lentiviral vectors targeting SLC6A1 , PCBP2, and PEX5L in renal medulla cells of camel in vitro with lentivirus‐NC/media = 1/5 (200×). Scale bar, 100 μm. (B) Expression levels of SLC6A1 , PCBP2, and PEX5L in the light of the ΔΔCt method in different groups. GAPDH was used as a loading control (intrinsic control), and expression levels were normalized with SLC6A1 , PCBP2, and PEX5L mRNA levels of NC and RNA interference groups. (C) Western blot targeting PCBP2. β‐actin was used as a loading control. (D) ROS detection after shRNA interference in renal medulla cells of camel by red fluorescent imaging DHE probe (100×). Scale bar, 100 μm. (E,F) Fluorescence intensity of ROS after shRNA interference against SLC6A1 , PCBP2, and PEX5L , and after small RNA interference against mixed SLC6A1 , PCBP2, and PEX5L (sRNA‐MIX). Data are presented as mean ± SD ( n = 3) by Student’s t‐test. Symbols: *** P < 0.001. shSLC6A1, shPCBP2, and shPEX5L represent short hairpin RNAs of SLC6A1, PEX5L, and PCBP2 in turn. IC refers to intrinsic control with GAPDH, NC indicates negative control with nonsense shRNA, and BC means blank control.

Article Snippet: SLC6A1, PCBP2, and PEX5L protein expression were severally examined using antibodies against‐SLC6A1 (ab426 and ab72448, Abcam, UK) and PCBP2 (ab96169, Abcam, UK) and PEX5L (nbp1‐91484, Novus Biologicals, USA).

Techniques: Transfection, shRNA, In Vitro, Expressing, Control, Western Blot, Imaging, Fluorescence, Negative Control

Journal: FEBS Open Bio

Article Title: Identification of sodium homeostasis genes in Camelus bactrianus by whole transcriptome sequencing

doi: 10.1002/2211-5463.13380

Figure Lengend Snippet: Salt‐resistance metabolism combined with competing endogenous RNAs (LNC003834, miRNA‐34a, and SLC14A1) and transcribed antioxidant genes ( SLC6A1 , PCBP2, and PEX5L ) in renal medulla of camel.

Article Snippet: SLC6A1, PCBP2, and PEX5L protein expression were severally examined using antibodies against‐SLC6A1 (ab426 and ab72448, Abcam, UK) and PCBP2 (ab96169, Abcam, UK) and PEX5L (nbp1‐91484, Novus Biologicals, USA).

Techniques:

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A , C Representative dorsal and ventral hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. B , D Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal and ventral hippocampi. E Western blot (top) and quantification (bottom) of HCN1 protein in dorsal CA1 region from the control, susceptible, and resilient groups. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly increased in dorsal CA1 neurons from susceptible group compared with those from the control and resilient groups. H – L We performed whole-cell voltage-clamp recordings. H Representative current responses with step voltage commands ranging from −140 mV to − 60 mV (Δ = 10 mV) at a holding potential of −60 mV. The approximate position for determining the peak tail current is shown by black vertical dashed lines. I Susceptible group showed increased I h in the dorsal CA1 neurons compared with those from the control and resilient groups. J The voltage dependence of activation for h channel was determined from tail currents ( I h / I h max ). The activation curve was fitted with a Boltzmann function with the following values: control V 1/2 = −101.8 mV, k = −14.13 mV, susceptible V 1/2 = −90.02 mV, k = −16.09 mV, resilient V 1/2 = −102.1 mV, k = −19.9 mV. K The half-activation voltage of h channel (V 1/2 ) for susceptible group was significantly shifted to the right by around +10 mV, whereas L the slope factor was not different between groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing, Western Blot, Activation Assay

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Illustration depicting the acute corticosterone treatment in the dorsal hippocampus. B Representative dorsal hippocampal slices immunolabeled with antibody against TRIP8b. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic TRIP8b protein expression. C Quantification of TRIP8b protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. D Representative dorsal hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. E Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly elevated in the dorsal CA1 neurons from corticosterone treatment compared with those from the vehicle-treated group. Dexamethasone reduced R in at RMP ( H ) and at −65 mV ( J ) in dorsal CA1 neurons. Corticosterone-induced decrease in R in at RMP ( H ) and at −65 mV ( J ) was blocked by RU 486, KT5720, and ZD7288. Changes in R in at RMP ( I ) and at −65 mV ( K ) were blocked by RU 486, KT5720, and ZD7288. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Timeline of CSDS, behavioral tests, electrophysiology, and biochemical assay. B CSDS produced the susceptible and resilient phenotype during the social interaction test. After 1 month ( C ) or 3 months ( D ) of no CSDS, susceptible mice showed persistent social avoidance during the social interaction test. E , F We performed whole-cell current-clamp recordings. E Representative voltage responses with depolarizing current step (210 pA; 750 ms) at RMP in dorsal CA1 neurons. F Dorsal CA1 neurons of susceptible group had lower action potential firing than control group, whereas the resilient group had higher action potential firing. G , H We performed cell-attached voltage-clamp recordings. G Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). H I h was significantly elevated in the dorsal CA1 neurons from susceptible group compared with those from the control and resilient mice. Representative dorsal hippocampal slices immunolabeled with antibody against HCN1 ( I ) and TRIP8b ( K ). Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 and TRIP8b protein expression. Quantification of HCN1 ( J ) and TRIP8b ( L ) protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. M , N We performed whole-cell current-clamp recordings. M Corticosterone reduced R in at RMP of the dorsal CA1 neurons in the control and resilient groups, but had no effect on R in in susceptible group. N Changes in R in at RMP were much lower in susceptible group compared to the control and resilient groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Produced, Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Illustration depicting the acute corticosterone treatment in the dorsal hippocampus. B Representative dorsal hippocampal slices immunolabeled with antibody against TRIP8b. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic TRIP8b protein expression. C Quantification of TRIP8b protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. D Representative dorsal hippocampal slices immunolabeled with antibody against HCN1. Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 protein expression. E Quantification of HCN1 protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. F , G We performed cell-attached voltage-clamp recordings. F Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). G I h was significantly elevated in the dorsal CA1 neurons from corticosterone treatment compared with those from the vehicle-treated group. Dexamethasone reduced R in at RMP ( H ) and at −65 mV ( J ) in dorsal CA1 neurons. Corticosterone-induced decrease in R in at RMP ( H ) and at −65 mV ( J ) was blocked by RU 486, KT5720, and ZD7288. Changes in R in at RMP ( I ) and at −65 mV ( K ) were blocked by RU 486, KT5720, and ZD7288. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Immunolabeling, Expressing

Journal: Molecular Psychiatry

Article Title: Glucocorticoid-glucocorticoid receptor-HCN1 channels reduce neuronal excitability in dorsal hippocampal CA1 neurons

doi: 10.1038/s41380-022-01682-9

Figure Lengend Snippet: A Timeline of CSDS, behavioral tests, electrophysiology, and biochemical assay. B CSDS produced the susceptible and resilient phenotype during the social interaction test. After 1 month ( C ) or 3 months ( D ) of no CSDS, susceptible mice showed persistent social avoidance during the social interaction test. E , F We performed whole-cell current-clamp recordings. E Representative voltage responses with depolarizing current step (210 pA; 750 ms) at RMP in dorsal CA1 neurons. F Dorsal CA1 neurons of susceptible group had lower action potential firing than control group, whereas the resilient group had higher action potential firing. G , H We performed cell-attached voltage-clamp recordings. G Representative maximal h current traces in response to a 500-ms hyperpolarizing voltage step (−140 mV). H I h was significantly elevated in the dorsal CA1 neurons from susceptible group compared with those from the control and resilient mice. Representative dorsal hippocampal slices immunolabeled with antibody against HCN1 ( I ) and TRIP8b ( K ). Rectangle boxes depict the region of the slice used for quantification of the optical density. The arrows indicate increased perisomatic HCN1 and TRIP8b protein expression. Quantification of HCN1 ( J ) and TRIP8b ( L ) protein expression from the perisomatic region to the distal dendritic region of CA1 from the dorsal hippocampus. M , N We performed whole-cell current-clamp recordings. M Corticosterone reduced R in at RMP of the dorsal CA1 neurons in the control and resilient groups, but had no effect on R in in susceptible group. N Changes in R in at RMP were much lower in susceptible group compared to the control and resilient groups. Data are expressed as mean ± SEM.

Article Snippet: Primary antibody in this study was used as follows; rabbit-anti-HCN1 (1:500, Invitrogen, Cat # PA5-78675), rabbit anti-HCN2 (1:500, Invitrogen, Cat # PA1-918), rabbit anti-HCN3 (1:500, Invitrogen, Cat # PA5-104434), rabbit anti-HCN4 (1:500, Invitrogen, Cat # PA5-111793), rabbit C-terminal TRIP8b (1:500, Proteintech, Cat #13084-1-AP), and rabbit anti-GR (1:500, Invitrogen, Cat # PA1-511A).

Techniques: Produced, Control, Immunolabeling, Expressing