Title Modified Western blotting for insulin and other diabetes-associated peptide hormones

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1 Supplemental Figures Title Modified Western blotting for insulin and other diabetes-associated peptide hormones Authors Naoyuki Okita, Yoshikazu Higami, Fumio Fukai, Masaki Kobayashi, Miku Mitarai, Takao Sekiya, Takashi Sasaki

2 Retrieval Order - Retrieval Quenching Quenching Retrieval Insulin Insulin Insulin Anti-insulin WB CBB (membrane) Supplemental Figure 1. Glycine quenching is effective when performed following the retrieval step. Dilution series (30, 100, 300 ng) of insulin were subjected to Tris/Tricine/urea SDS-PAGE. After electro-blotting/ga fixation, the one blotted membrane was separated into 3 slips for the control experiment (not subjected to retrieval and quenching steps) and the described experiments. To compare the effects of various conditions, WB images in each sub-part of the figure were captured at one time.

3 General procedure Proinsulin /Insulin Improved procedure Proinsulin /Insulin Anti-insulin Supplemental Figure 2. The improved methods are also effective when using another antibody against insulin (clone L6B10). Dilution series of equal moles of proinsulin and insulin (range, 51.1 to 0.5 pmol) were subjected to the general or the improved WB procedure with anti-insulin clone L6B10. The blotted slips for the general or improved protocol were separated from one blotted membrane.

Proinsulin /Insulin [fmol] Serum Anti-insulin Supplemental Figure 3. Application of the improved method for mouse serum sample. (a) Proinsulin/insulin standards were used at a range of 155-15.

4 Proinsulin /Insulin [fmol] Serum Anti-insulin Supplemental Figure 3. Application of the improved method for mouse serum sample. (a) Proinsulin/insulin standards were used at a range of fmol (Proinsulin, pg; Insulin, pg). As a serum sample, 1 µl of pooled serum (n=9) from normal diet-fed or high fat diet-fed C57BL/6 mice (both sexes, 22 weeks old) was prepared. Blood sampling was performed under no regulation of food intake. The samples were subjected to the improved WB.

5 a Insulin standards Spike [ng] Cell lysate Recovery rates 200 ng 105.0±5.6% 50 ng 124.4±4.4% b Insulin standards Spike [ng] Medium Recovery rates 25 ng 125.8±2.6% 6.25 ng 125.9±13.8% Supplemental Figure 4. Spike and recovery experiment in the improved method. Spike and recovery experiments were performed using MIN6c4 cell lysate (a) or culture medium (b). (a) Insulin standards were used at a range of ng (each 1/2 dilution, 6 points). As a cell lysate sample, 5 μg of extracted protein of MIN6c4 cells maintained with DMEM containing 12.5 mm glucose was prepared. Insulin spiking doses to cell lysates were 200 or 50 ng. (b) Insulin standards were used at a range of ng (each 1/2 dilution, 6 points). As a medium sample, a culture supernatant from MIN6c4 cells incubated with KRBH buffer containing 30 mm glucose for 2 h as shown in Methods was prepared. Insulin spiking doses to medium were 25 or 6.25 ng. The shown recovery rates (±standard error) were represented by averages from 4 independent data. The representative images were shown.

Supplemental Figure 5. WB of somatostatins in pancreas and islet sample. Somatostatins in mice pancreas and islet lysate (5 μg) were analyzed by WB with or without the additional steps.

6 Supplemental Figure 5. WB of somatostatins in pancreas and islet sample. Somatostatins in mice pancreas and islet lysate (5 μg) were analyzed by WB with or without the additional steps. WB images in each sub-part of the figure were captured at one time. U.I.S. means unidentified signals. CBB staining of the blotted membrane were performed to ensure appropriate loading. The loaded standards of somatostatins were as follows: somatostatin-28/somatostatin-14, 611 to 6.11 fmol (each 1/10 dilution, 3 points).

7 Supplemental Information: Full Blot Images of WB in the Manuscript Title Modified Western blotting for insulin and other diabetes-associated peptide hormones Authors Naoyuki Okita, Yoshikazu Higami, Fumio Fukai, Masaki Kobayashi, Miku Mitarai, Takao Sekiya, Takashi Sasaki Description In the supplemental information, the original images (monochrome inversion) are shown without auto-contrast adjustment of Adobe photoshop software. Seven-digit numbers described after figure numbers, the data acquisition date (YYYYMMDD): Squares with bold solid lines, the location of images used in the main figures: Dot lines, the location of borderline between the different membranes captured at one time: Digital 6 dots traced in the WB images, the location of the colored molecular weight markers ( 42, 30, 16, 10, 4.6, and 1.7 kda) (CST Cat. No.13070).

Fig.1 (20150217) SDS conc. in sample buf.

5 0.5 2.5 0.5 2.5 0.5 2.5 0.5 2.5 SDS conc.

8 Fig.1 ( ) SDS conc. in sample buf. [%] Insulin [µg] SDS conc. in running buf. : 0.1% SDS conc. in running buf. : 0.05%

Fig.2a (20150328-1) PIns Ins [ng] Anti-Insulin WB

9 Fig.2a ( ) PIns Ins [ng] Anti-Insulin WB Proinsulin Insulin CBB staining after WB (membrane) Proinsulin

10 Fig.2b ( ) PIns Ins [ng] CBB staining immediately after blotting (membrane) CBB (gel)

11 Fig.3a ( ) - Paraformaldehyde [%} Anti-Insulin b chain WB Membrane CBB

12 Fig.3b ( ) - Glutaraldehyde [%} Anti-Insulin b chain WB Membrane CBB

Fig.3c (20150422) GA - + Retrieval - - + + Anti-Insulin b

13 Fig.3c ( ) GA - + Retrieval Anti-Insulin b chain WB Proinsulin Insulin Membrane CBB Proinsulin Insulin

14 Fig.4a, b ( ) High dose(100-3 ng) Low dose( ng) NT Blocking x1/100 NT Blocking x1/100 Blocking x1/10 Blocking x1 Blocking x1/10 Blocking x1

15 Fig.4c ( ) Low dose( ng) 5 min 10 min 30 min

16 Fig.5a ( ) ( pmol) General procedure Improved procedure Insulin Proinsulin Insulin

17 Fig.5g ( ) ( pmol) Anti-Insulin WB Proinsulin Insulin CBB(membrane)

Fig.5h (20161123) Pro(215.24 3.36 fmol) Ins(2152.4-33.

18 Fig.5h ( ) Pro( fmol) Ins( fmol) Anti-insulin WB Proinsulin Insulin CBB(membrane)

19 Fig.6a ( ) Anti-Insulin - 0.2% GA 0.4% PFA

20 Fig.6b ( ) Anti-Glucagon - 0.2% GA 0.4% PFA

21 Fig.6c ( ) Anti-GLP-1-0.2% GA 0.4% PFA

22 Fig.6d ( ) Anti-somatostatin - 0.2% GA 0.4% PFA

23 Fig.6e ( ) Anti-Ghrelin - 0.2% GA 0.4% PFA

24 Fig.6f ( ) Anti-Pancreatic polypeptide - 0.2% GA 0.4% PFA

25 Fig.7a ( ) pmol Anti-insulin proinsulin insulin CBB

26 Fig.7b ( ) pmol Anti-glucagon Unidentified bands glucagon CBB

27 Fig.7c ( ) pmol Anti-GLP-1 GLP-1(1-36)amide Unidentified bands GLP-1(7-36)amide CBB

28 Fig.7d ( ) pmol pmol Anti-insulin CBB - 0.2% GA

29 Fig.7e ( ) pmol pmol Anti-glucagon CBB - 0.2% GA

30 Fig.7f ( ) pmol pmol Anti-GLP-1 CBB - 0.2% GA

mm Distance (mm)

mm Distance (mm) b a Magnet Illumination Coverslips MPs Objective 2575 µm 1875 µm 1575 µm 1075 µm 875 µm 545 µm 20µm 2 3 0.5 0.3mm 1 1000 100 10 1 0.1 1000 100 10 1 0.1 Field Induction (Gauss) 1.5 0 5 10 15 20 Distance